3377 The Journl of Experimentl iology 212, 3377-3386 Pulished y The Compny of iologists 29 doi:1.1242/je.25221 The role of experience in flight ehviour of Drosophil Thoms Hesselerg nd Fritz-Olf Lehmnn* iofuture Reserch Group, Institute of Neuroiology, University of Ulm, lert-einstein-llee 11, 8981 Ulm, Germny *uthor for correspondence (fritz.lehmnn@uni-ulm.de) ccepted 21 July 29 SUMMRY Experience plys key role in the cquisition of complex motor skills in running nd flight of mny vertertes. To evlute the significnce of previous experience for the efficiency of motor ehviour in n insect, we investigted the flight ehviour of the fruit fly Drosophil. We rered flies in chmers in which the nimls could freely wlk nd extend their wings, ut could not gin ny flight experience. These nive nimls were compred with control flies under oth open- nd closed-loop tethered flight conditions in flight simultor s well s in free-flight ren. The dt suggest tht the overll flight ehviour in Drosophil seems to e predetermined ecuse oth groups exhiited similr men stroke mplitude nd stroke frequency, similr open-loop responses to visul stimultion nd the immedite ility to trck visul ojects under closed-loop feedck conditions. In short free flight outs, pek sccdic turning rte, ngulr ccelertion, pek horizontl speed nd flight ltitude were lso similr in nive nd control flies. However, we found significnt chnges in other key prmeters in nive nimls such s reduction in men horizontl speed ( 23%) nd sutle chnges in men turning rte ( 48%). Nive flies produced 25% less yw torqueequivlent stroke mplitudes thn the controls in response to visul stripe rotting in open loop round the tethered niml, potentilly suggesting flight-dependent dpttion of the visuo-motor gin in the control group. This chnge cesed fter the nimls experienced visul closed-loop feedck. During closed-loop flight conditions, nive flies hd 53% lrger differences in left nd right stroke mplitude when fixting visul oject, thus steering control ws less precise. We discuss two lterntive hypotheses to explin our results: the neuronl experience hypothesis, suggesting tht there re some elements of lerning nd fine-tuning involved during the first flight experiences in Drosophil nd the musculr exercise hypothesis. Our experiments support the first hypothesis ecuse mximum locomotor cpcity seems not to e significntly impired in the nive group. lthough this study primrily confirms the genetic pre-disposition for flight in Drosophil, previous experience my pprently djust locomotor fine control nd eril performnce, lthough this effect seems to e smll compred with vertertes. Key words: motor lerning, locomotor plsticity, flight experience, free-flight performnce, sccdes, wing kinemtics, force production, fruit fly. INTRODUCTION The study of niml ehviour differentites etween two types of ehviours: innte geneticlly determined ehviourl sequences, so-clled stereotypic ehviour or fixed-ction ptterns, nd cquired or lerned ehviour. During the erly hlf of the lst century, the field of ethology ws dominted y the theory of instinctive ehviour proposed first y Lorenz (Lorenz, 1937) nd lter supported y Tinergen (Tinergen, 1951), which climed tht most of the oserved ehviours in nimls were instinctive nd therefore not influenced y experience. However, the theory of instinctive ehviour ws soon recognised s eing too simplistic, t lest for vertertes where lrge prt of ehviour rises from interctions with the environment nd with conspecifics (Lehrmn, 1953). Thus tody, it is recognised tht the cquisition of complex motor skills in vertertes results from the interply etween the physiologicl development of the locomotor pprtus nd long-lsting motor prctice. In humns it typiclly tkes severl yers of prctice to ecome skilled in dexterous tsks nd sport disciplines, nd irds lern to fly efficiently within dys or weeks fter leving the nest (Yod et l., 24; Hmer et l., 22). Trditionlly, flight in irds is ssumed to e correlted with mturtion nd thus with the development of muscles nd neurons, without ny lerning tking plce (Grohmnn, 1939). recent study on the rown ooy Sul leucogster, however, showed tht flight durtion decreses nd the proportion of time spent gliding increses, with the numer of dys since fledging, indicting tht flight ecomes more efficient with experience in this seird (Yod et l., 24). Motor skills in insects nd other invertertes, y contrst, re still widely recognised s eing predominntly innte fixed-ction ptterns. Evidence for this view comes from lrge ody of oservtions on wide group of nimls. Or spiders, for exmple, uild their wes y following stereotypic set of rules without relying on ny previous we-uilding experience (Witt et l., 1972). Other exmples include the stereotypic set of locomotor ctions used y mny wlking insects, such s stick insects (lesing nd Cruse, 24) nd fruit flies (Pick nd Struss, 25), when they encounter nd cross gps, the initition of flight in locusts when sensory hirs on the hed detect hed wind (Wilson, 1961) nd the lnding response in flies occurring in response to expnding visul flow fields (orst, 199). The improvement in flight cpcity of utterflies during the first dys fter emergence, y contrst, results from cuticle hrdening nd not from motor lerning (Petersen et l., 1956). During the pst few decdes severl studies hve repetedly emphsized tht these stereotypic ehviourl ptterns yield some degree of plsticity nd re potentilly shped y previous experiences. Spiders, for exmple, mke smll chnges to their weuilding routine resulting in functionl chnges in the geometry of their wes in response to lrge set of environmentl nd physiologicl fctors including previous prey experiences (Venner et l., 2). Similrly, the jumping spider Porti fimrit shows tril-nd-error lerning, in which previous success of motor
3378 T. Hesselerg nd F. O. Lehmnn ehviours, such s the escpe from islnds y jumping or swimming, determines future ctions in similr context (Jckson et l., 21). Insects lso redily modify stereotypic motor sequences s result of sensory input. Tethered locusts experience n immedite decrese in wing stroke frequency fter ltion of hind wing proprioceptors ut they regin the initil frequency within dys, when the insects compenste for the loss y relying on other proprioceptors (Gee nd Roertson, 1996). In nother study, experimentl mnipultions of the feedck loop in tethered locusts showed tht these nimls dpt to n rtificilly creted symmetry etween the wing steering muscles nd tht this dpttion is rememered in susequent open-loop flight (Möhl, 1988). Locusts lso seem to possess n internl representtion of the movement of their hind legs during scrtching movements tht remins fixed to ody coordintes (Mtheson, 1997). Flies disply remrkle repertoire of complex erotic ehviours including hovering, trcking, collision nd escpe responses, nd n elorte lnding progrmme medited y visul pthwys nd hltere feedck (Frye, 27; Tmmero nd Dickinson, 22; Wgner, 1986). Forwrd flight is, furthermore, chrcterised y stereotypic rpid sccdes in which the fly chnges yw heding etween 9deg. nd 12deg. in 5 13ms (Mronz nd Lehmnn, 28; Fry et l., 23; Tmmero nd Dickinson, 22; Myer et l., 1988). Thus, efficient flight in Drosophil requires sustntil motor skills s well s strong coordintion etween sensory input nd motor output. To our knowledge no previous studies hve investigted to wht degree flight ehviour in these flies, or indeed in ny other insect, is cquired during postpupl development. Recent studies, however, demonstrte tht there is plsticity nd lerning in the motor system of dult fruit flies. Wng et l. (Wng et l., 23), for exmple, found tht the nimls lern to modulte their torque output in response to visul pttern when trined with het punishment. Fruit flies re, moreover, le to compenste for wing dmge nd remin irorne despite hving up to hlf of their wings removed (ender nd Dickinson, 26). In this study we investigted the question of whether flight of the fruit fly is purely innte or improves with prctice. We ddressed this question y compring vriety of flight prmeters such s wing kinemtics, flight speed nd ccelertion, yw torqueequivlent stroke mplitude responses during oject trcking, nd sccdic turning ngles nd rtes etween experienced nd nive flies without previous flight experience. This ws done under oth tethered flight conditions in simultor nd free-flight conditions in free-flight ren. MTERILS ND METHODS nimls The tested flies were selected from lortory colony, mintined t 24 C on commercil Drosophil feed (Crolin iologicl, urlington, NY, US). Two dys efore htching, five pupe were crefully trnsferred, without directly touching the pupe, into stndrd 35 mm dimeter Drosophil vil (control nimls) nd nother five into 8mm 6mm 2mm (length width height) custom-uilt flt Perspex rering chmer (nive nimls, Fig. 1). oth vils were kept under identicl conditions in reeding chmer under 12h:12h light:drk cycle. In oth chmers htched flies hd ccess to food offered in 35 mm dimeter circulr continer. Free wlking re ws not very different etween the two chmers, mounting to pproximtely 479 nd 984mm 2 for the stndrd vil nd the custom-uild chmer, respectively. y contrst, free totl spce ws lrger in the stndrd (37 1 3 mm 3 ) thn in the custom-uilt chmer (9.6 1 3 mm 3 ). Owing to the low popultion density nd the reltively lrge surfce re of oth chmers, the flies could redily wlk round, crry out courtship ehviour nd lso mte. lthough we cnnot completely exclude sutle chnges, we did not oserve differences in the ehviours etween the two groups, except tht the flt chmer did not llow the nive group to gin flight experience. Oservtions of undistured flies in the stndrd vils showed tht they initited short flight outs, thus giving them the chnce of free flight prior to our experiments. We did not record flight time nd distnce during the flight outs in the controls, thus it is possile tht these flies hd some vritions in their ctul flight prctice prior to testing. ll flight experiments were crried out on 4- to 5-dy-old femle Cnton S wild-type Drosophil melnogster Meigen. close visul inspection of the nimls using imge nlysis softwre (Scion Imge, Frederick, MD, US) reveled no mjor morphologicl differences etween the two groups. Thorx width, mesured etween left nd right frontl notopleurl sete mounted to.738±.6 mm nd.745±.47 mm in control nd nive flies, Flt running chmer Food continer IR-diode Flight ren Holder C High-speed cmer Sttionry cylinder FLT se plte Cmer Pttern cylinder Wing stroke nlyser Motor Fig. 1. Experimentl setups. () Rering chmer for nive fruit flies, consisting of se plte nd rectngulr running chmer with circulr food supply in the middle. () Virtul-relity flight simultor. The tethered flies ctively control the zimuth velocity of the visul oject (lck stripe) y modulting the difference of their wing stroke mplitudes. mplitudes re mesured y n infr-red light pth (red) tht csts shdows of the eting wings on wing stroke nlyser. n infr-red cmer simultneously trcks the wings for clirtion. (C) Free-flight ren. Single flies emerge in the middle of the ren on n elevted pltform nd voluntrily initite flight. high-speed video cmer is triggered when the niml tkes off, nd crdord shields the experimentl setup from mient light. The ger motor rottes the rndom-dot visul environment nd three circulr fluorescent light tues (FLT) illuminte the visul pttern from ehind. Drwings re not to scle.
Significnce of experience in flight ehviour 3379 respectively (N=16, two-tiled t-test on mens, P=.67), nd thorx length, mesured etween the most frontl row of hirs on the thorx nd the tip of the scutellum ws.939±.62mm nd.951±.61mm for the two groups, respectively (mens ± s.d.). Similr results were otined for wing length (controls: 2.35±.8 mm, N=19; nive nimls: 2.37±.14 mm, N=33; two-tiled t-test on mens, P=.7) nd wing re (controls: 1.77±.14mm 2, N=19; nive nimls: 1.79±.21mm 2, N=33; two-tiled t-test on mens, P=.7). lthough ody mss ws not significntly different etween the two groups (controls: 1.12±.25 mg, N=36; nive nimls: 1.6.37±.22 mg, N=6; two-tiled t-test on mens, P=.25), control flies tended to e hevier thn the nive nimls. ltogether, the ove vlues suggest no mjor differences in flight muscle volume, power requirements for lift production nd erodynmic effects s result of wing size for the two groups. Flight simultor The flight simultor used in this study hs lredy een descried in detil y Lehmnn nd Dickinson (Lehmnn nd Dickinson, 1997), so only rief introduction is given here. The tungsten rod on the tethered flies fitted into holder tht plced the fly in the middle of cylindricl flight simultor, 125mm high nd 15mm in dimeter (Fig.1). The inclintion of the holder ensured tht the fly ws in hovering position, with ody ngle of 6deg., so tht the stroke plne of the wings coincided with the horizontl (Dvid, 1978). n infr-red diode ove the flight simultor cst shdows of the wings on n infr-red-sensitive msk connected to wing stroke nlyser tht provided wing stroke mplitudes nd frequency for ech single stroke cycle. We clirted wing stroke mplitudes y digitizing the wing positions on video imges of the flying niml recorded y n infr-red-sensitive cmer. The voltges coming from the infr-red light pth were susequently converted into degrees using liner regression on the digitised dt. oth digitistion nd the finl clirtion were done with custom-uilt Origin (Version 7, OriginL corportion, M, US) routines. conventionl computer generted 12 deg. wide lck r foreground on uniform green ckground (Fig. 1). The imge displyed in the simultor ws updted every 8 ms nd flickered with frequency of 1 Hz, which is well ove Drosophil s flicker fusion rte of round 2 Hz (utrum, 1958). The pttern could move in oth n open loop, where the stripe rotted uniformly t speed of 36deg.s 1, independent of the fly s ehviour, nd in closed loop, where the fly ctively controlled the zimuth velocity of the pttern y chnging the reltive difference of the stroke mplitude etween oth wings (left minus right). Throughout the mnuscript, vlue of deg. stripe position indictes the frontl zimuth position of the compound eyes, wheres 9 deg. (9 deg.) ngle indictes the lterl position on the left (right) ody side. The reltionship etween ngulr velocity of the imge nd the wing stroke difference ws controlled y physics engine tht simulted physicl conditions similr to free flight such s the frictionl dmping on ody nd wings nd inertil moments of the fly. In ll experiments we used dmping coefficient of 52 1 12 Nms nd n inertil coefficient of.52 1 12 Nms 2, ecuse previous study showed tht these vlues yielded the most precise visul control feedck of tethered Drosophil (Hesselerg nd Lehmnn, 27). Free-flight ren The free-flight ren consisted of two concentric, 2 mm high crylic cylinders with 14 mm nd 19 mm dimeter, respectively [for detiled description see Mronz nd Lehmnn (Mronz nd Lehmnn, 28)] (Fig. 1C). The inner trnslucent cylinder ws immovele nd prevented the flies from lnding on the second outer cylinder on which visul 8deg. 8deg. wide rndom squre pttern ws ttched. Three surrounding circulr fluorescent light tues illuminted the pttern cylinder from outside, while the cylinder s frosted surfce estlished diffusive nd lmost uniform light conditions within the ren. When flies took-off from the centrl pltform, high-speed cmer recorded the flight pth of the fly from ove t 125 frmes per second with resolution of 12 12 video pixels. For ech niml we recorded n 8s sequence using Pixoft video cpturing softwre (Pixoft V3., irminghm, UK). Since we imed to evlute the flies t their mximum locomotor cpcity, the outer pttern cylinder rotted t 5deg.s 1 counter-clockwise to elicit visul optomotor ehviour (Mronz nd Lehmnn, 28). We strted the rottion efore the fly took-off so tht uniform speed of the cylinder ws chieved when the insect initited flight ehviour. We nlysed the recorded video imges using custom-uilt softwre written in Visul C (Microsoft, Redmond, US) nd commercil imging components (Mtrox Imging Lirry Mil 7.5, Dorvl, Cnd), extrcting dt on (i) the position of the fly s centre of grvity, (ii) the ngulr orienttion of the longitudinl ody xis, nd (iii) the size of the fly using lo nlysis from ech frme. These dt were susequently converted into (i) totl flight distnce covered, (ii) lterl distnce to the ren wll, (iii) flight ltitude, (iv) horizontl velocity nd (v) ccelertion, nd finlly the niml s turning (ngulr) velocity round the yw xis. Flight ltitude ws estimted from liner regression [y= 731.5x, where x is the lo size in pixel (Mronz nd Lehmnn, 28)] nd used to correct the fly s x/y position to otin exct velocity estimtes. Similr to previous studies, we scored flight mnoeuvres s sccdes when the turning rte of the niml exceeded 1deg.s 1. From yw orienttion of the fly s longitudinl ody xis, we estimted the following sccde prmeters: (i) rottionl direction, (ii) totl turning ngle, (iii) sccde frequency nd (iv) ngulr velocity during the sccde. Experimentl procedures The flies were trnsported in smll vils nd relesed individully into tue leding to the centre of the free-flight ren y mens of microprocessor-controlled gte. The nimls voluntrily wlked through the tue nd into the ren nd usully took off within second fter emerging in the centre. y contrst, the flies used in the flight simultor were nesthetised y cooling them to pproximtely 4 C nd tethered etween the hed nd the notum to 7.3mm long,.13mm dimeter tungsten rod using UV-light ctivted glue. The flies were tken to the flight simultor immeditely fter tethering in which they were llowed to recover. Only 13% of the controls (3 of 23) nd 14% (5 of 36) of the nive nimls did not initite flight voluntrily within 3 min time period fter moving. In these cses, we initited flight y gently lowing ir on the niml s wings. Men recovery time ws similr in oth groups (controls: 16.5±1.5 min, N=19; nive nimls: 2.8±11.3 min, N=33; two-tiled t-test on mens, P=.18). The experiment strted s soon s the nimls initited wing movements. Ech simultor experiment ws 22 s long nd divided into the following flight sequences: (i) n initil open-loop feedck sequence of 2s split into 1s, in which the stripe rotted one full circle in the clockwise direction nd further 1s, in which it rotted full circle in the counter-clockwise direction, (ii) 18s of closedloop feedck nd (iii) finl open-loop feedck sequence similr to the initil score (Fig. 2). In the open-loop sequences the stripe lwys ppered directly ehind the fly nd then moved into its visul field. Flies tht hd n intermedite flight stop in one of the
338 T. Hesselerg nd F. O. Lehmnn Stripe position (deg.) Open-loop cw ccw 2 s 2 2 s 2 22 s 2 1 1 Closed-loop 2 5 1 15 2 25 Time (s) Open-loop cw ccw Fig. 2. Experimentl procedure. The flight sequence consisted of n initil 2 s open-loop sequence in which single stripe rotted full cycle clockwise (cw, green) nd counter clockwise (ccw, red), in ech cse strting from position in the rer ( 18/18 deg.). The ngulr velocity of the stripe ws 36 deg. s 1. During the 18 s closed-loop sequence, the fly ctively controlled the zimuth position of the stripe in the frontl field of view. The finl 2 s open-loop sequence ws similr to the first. Dt re tken from the flight of single control niml. sequences were excluded from the nlysis. We found no mjor difference in the reltive numer of exclusions etween control nd nive flies, suggesting tht oth groups hd similr motivtion for flight (controls: 42%, nive nimls: 34%). In generl, we crefully ensured tht nive flies were given no opportunity to fly efore they hd een tested either in the flight simultor or in the free-flight ren. mient temperture ws similr in the experiments; 22.9±1.32 C (control) nd 23.3±1.36 C (nive; mens ± stndrd devition, two-tiled t-test, P=.28). Sttistics In open-loop conditions, we found distinct correltion etween stripe position nd the difference in stroke mplitude etween oth wings, s expected from previous work (Heisenerg nd Wolf, 1984). For further comprison, we thus fitted oltzmnn wve to the individul responses within ±12 deg. visul rnge using the oltzmnn fitting function in Origin, which is sed on the simplex serch method (Lgris et l., 1998). To test for chnges in the ngulr velocity profile during flight sccdes, we clculted the mens derived from dt pooled from 8ms efore to 8ms fter pek turning velocity. For sttisticl tests we used SPSS (version 1., SPSS, 1999) nd Origin t significnce level of 5%. If not stted otherwise, dt re given s mens ± stndrd devition. RESULTS Tethered flight experiments fter eing plced in the ren, the mjority of flies voluntrily initited flight ehviour nd ttempted to fixte the stripe in the frontl region of their compound eyes. We nlysed the ehviourl dt seprtely for the two open-loop nd the longer closed-loop sequences to highlight the following spects. First, dt from the initil open-loop sequence ( 2s) llowed us to determine the initil stroke mplitude-to-stripe position representtion in the two groups nd thus to estimte the effect of previous flight experiences. In this nlysis, we evluted wing kinemtics nd flight force production. Second, during closed-loop (2 2 s) flight we determined wing kinemtics nd fixtion ehviour towrds the lck stripe in order to identify ny potentil differences in the ility of the two groups to visully control the zimuth velocity of the oject. Third, we used the dt recorded in the finl open-loop sequence (2 22s) to highlight potentil chnges in the flies internl gin for stroke mplitude-to-stripe position, derived from the previous closed-loop experience. Fig. 3 shows typicl time trces of the kinemtic mplitude difference (deg.) Men mplitude (deg.) Stroke frequency (Hz) 8 4 4 8 18 16 14 12 24 C D 22 2 18 2 2 22 2 2 22 Time (s) E F Fig. 3. Flight dynmics in tethered flight. The left column ( C) shows wing kinemtics for n experienced fly, the column on the right (D F) for nive fly. The upper row shows the difference in wing stroke mplitude etween the left nd the right wing tht is equivlent to yw torque. Lift on the fly ody is proportionl to the product etween men stroke mplitude (middle row) nd stroke frequency (lower row). The grey res indicte open-loop (OL) flight; closed-loop (CL) flight. Grphs show rw mesurements (grey dots) nd temporlly filtered responses (red, lue).
Significnce of experience in flight ehviour 3381 prmeters for the three flight conditions: the reltive difference etween left nd right stroke mplitude, men stroke mplitude of oth wings nd stroke frequency, for n experienced (Fig. 3 C) nd nive fly (Fig.3D F). Tethered flight: open-loop conditions During open-loop flight, when the stripe rotted t constnt speed cross the flies visul field, the flies typiclly steered towrds the stripe y chnging the reltive difference etween left nd right stroke mplitude, producing sinusoidl response curve (Fig. 4). Men stroke mplitude nd stroke frequency, y contrst, were less dependent on stripe position. We found only smll, insignificnt chnges in men mplitude difference etween experienced nd nive flies in oth open-loop sequences (t-test, 2 s: P=.59; 2 22s: P=.65; Fig.4, inset). The sme holds for men stroke mplitude (t-test, 2 s: P=.23, 2 22 s: P=.35; Fig. 4, inset) nd stroke frequency (t-test, 2 s: P=.75, 2 22 s: P=.96; Men mplitude (deg.) mplitude difference (deg.) Stroke frequency (Hz) 3 15 15 3 165 16 155 15 145 25 2 195 C 5 deg. 1 deg. 1 Hz 19 18 12 6 6 12 18 Stripe position (deg.) Fig. 4. Men wing stroke responses of experienced (red, ) nd nive flies (lue, ) during the initil open-loop sequence ( 2 s), plotted s function of stripe position. () Difference etween left nd right stroke mplitude, () hlf sum stroke mplitude of oth wings nd (C) stroke frequency. Insets (grey columns) show men vlues nd stndrd devitions of the prmeters in the open-loop sequence. Men stndrd devition of ech curve is: 14.8 deg. (,), 19.7 deg. (,), 12.4 deg. (,), 11.3 deg. (,), 13.9 Hz (C,) nd 18.6 Hz (C,). N=18 nd 32 for experienced (control) nd nive flies, respectively. Fig. 4C, inset). In ddition, we found no significnt difference for ny of the three kinemtic prmeters etween the two open-loop flight sequences [t-test, control (nive), stroke mplitude difference: P=.57 (.77), men mplitude: P=.12 (.7), frequency: P=.87 (.58)]. To sttisticlly evlute the mplitude response to the moving stripe, we fitted oltzmnn wve to the mplitude difference etween ±12 deg. of the visul field nd scored oth the totl ngulr response ( P ; Fig.5) nd the offset of the fit curve (d F ; Fig.5). Fig.5 shows tht the response mplitude in the nive flies ws 29.4±13.7deg. (N=32 flies) nd thus significntly smller thn the controls (39.1±14.8 deg., N=18, t-test, P=.3). For the finl open-loop sequence (2 22 s flight time), we otined slightly smller vlues, ut did not find significnt difference etween those mesures (control: 25.±17. deg., nive: 22.8±17.4 deg., t- test, P=.66). The curve offset (d F ) ws not sttisticlly different etween control nd nive nimls or etween oth open-loop sequences (t-test, P>.5; Fig. 5). possile explntion for the decrese in response mplitude during open-loop flight of nive flies is reduction in the nimls cpcity to produce elevted flight forces. However, this seems not to e the cse. We derived totl flight force production from the squred product etween men stroke mplitude nd stroke frequency using conventionl qusi-stedy pproch (Ellington, 1984). Since men lift coefficient, C L, in tethered flight depends on men stroke mplitude,, we djusted this prmeter ccordingly [C L = 5.4643.5 1 3 (Lehmnn nd Dickinson, 1998)]. Men totl flight force verged over the entire 22s flight time ws similr in oth experimentl groups (control: 7.97±2.55 μn, nive: 7.6±1.99 μn, t-test, P=.17) including pek force (mximum 5% of ll force dt) during open-loop (two-tiled t-test, 2 s: P=.66, 2-22 s: P=.37; Fig. 6, inset) nd closed-loop (control: 11.1±3.24μN, nive: 1.±3.22μN, t-test, P=.24) flight. Moreover, we did not find ny correltion etween response mplitude nd pek force production in oth open-loop sequences of control nimls (liner regression fit, 2 s: y=49.8.81x, R 2 =.6, P=.31, N=18 flies; 2 22s: y=9.32.97x, R 2 =.16, P=.8, N=32 flies) nd nive flies (liner regression fit, 2 s: y=35.3.41x, R 2 =.2, P=.46, N=2 flies; 2 22 s: y=11.11.3x, R 2 =.8, P=.13, N=32 flies; Fig.6), ltogether suggesting tht response mplitude nd thus mximum yw torque s shown in Fig. 5 is independent of the niml s mximum locomotor cpcity. Tethered flight closed-loop conditions While fixting the lck stripe under closed-loop visul feedck condition in the frontl region of their visul field, oth groups exhiited similr men stroke mplitude (control: 151±11.7 deg., N=2 flies; nive: 149±9.84 deg.; N=3 flies; t-test, P=.4) nd lmost identicl stroke frequencies (control: 26±11.4, nive: 25±15.3 Hz, t-test, P=.95). However, similr to wht hd een mesured during the open-loop sequences, we found sutle chnges in steering control ehviour etween the two groups. The exmples in Fig.7 show tht nive flies used more corrective steering when fixting the stripe, which resulted in slight shift in the fst Fourier trnsformed (FFT) spectrum of left-minus-right stroke mplitudes towrds higher frequencies ove pproximtely 2 Hz nd pronounced decrese in FFT mplitude t frequencies elow this vlue (Fig. 8). On verge, nive flies exhiited 53% higher solute mplitude difference etween wings (control: 2.3±1.3 deg., N=2; nive: 4.84±1.57 deg., N=3; t-test, P<.1) during closed-loop flight thn the controls. Consequently, stripe fixtion ehviour ws less precise in nive flies s shown y the
3382 T. Hesselerg nd F. O. Lehmnn Response difference (deg.) 5 25 P d F 25 5 18 12 6 6 12 18 Stripe position (deg.) Response difference (deg.) 6 45 3 P=.1 P=.3 P 15 2 s 2 22 s df 2 s 2 22 s Fig. 5. Response mplitudes during open-loop flight in experienced nd nive fruit flies. () The difference (L R) etween left (L) nd right (R) stroke mplitude chnges with the ngulr position of the lck stripe displyed in the ren. Dt were recorded during the first open-loop sequence ( 2 s). Ech dt point represents the verge of two mesurements derived during clockwise nd counter-clockwise rottion of the stripe (cf. Fig. 2). The fly s internl torque-position representtion ws clculted from the mplitude response ( P ) of fitted oltzmnn curve (red). d F, offset of oltzmnn fit curve. () mplitude difference during the initil 2 s (,) nd finl 2 22 s (, ) open-loop flight sequence. Vlues re mens ± s.d.;, not significnt (ttest). mens of solute stripe position (control: 26.1±14.7 deg., N=2; nive: 37.2±7.73 deg., N=3; t-test, P=.1, 2 2s flight). Nevertheless, the overll differences etween oth niml groups were reltively little, gin supporting the ssumption of geneticlly pre-progrmmed locomotor pprtus. Free-flight ehviour Since potentil differences in eril performnce should e most visile t elevted motor ctivity of the nimls, when most of the locomotor reserves re needed, we compred flight of the two groups in free-flight ren rotting t 5deg.s 1 (see Mterils nd methods). Previous dt hve lredy shown tht in Drosophil flying in sttionry ren, flight speed nd turning rte is typiclly 4 nd 7% elow the performnce mesured during optomotor response, respectively (Mronz nd Lehmnn, 28). In this regrd, we here score the nimls t extreme situtions, comprle, for exmple, to escpe mnoeuvres from eril predtors in the wild. The flies initited flight lmost s soon s they hd entered the ren nd followed the rotting pnorm on circulr flight pths (Fig.9 F). We oserved no mjor differences etween nive nd experienced flies in tke-off ltency or trnsfer proility inside the ren (Fig. 9G,H). Nive flies, however, flew pproximtely 23% slower thn experienced flies nd thus covered significntly shorter distnce (Tle1). Despite the lower flight speed, solute horizontl ccelertion ws 3.3 times higher in the nive nimls wheres pek cruising speed ws not significntly different etween the two groups (.84 vs.73ms 1 ). Flight y the nive flies ws thus either more errtic or mnoeuvrle, with higher fluctution in forwrd speed. Response mplitude (deg.) 8 6 4 2 8 6 4 2 Nive Control 5 µn Control Nive 5 µn 5 1 15 2 25 Pek force (µn) Fig. 6. Response mplitude (difference in stroke mplitude) plotted ginst mximum force production. () mplitudes of oltzmnn fitted curve (cf. Fig. 5) during the initil ( 2 s) nd () finl (2 22 s) open-loop sequence. Mximum force vlues represent the men of the upper 5% force vlues within ech flight sequence. Insets show verged pek force vlues nd stndrd devitions for control (red, ) nd nive (lue, ) nimls. N=18 (control, ), 2 (control, ), 32 (control, ) nd 28 (nive flies, ). For clcultion of totl flight force, see text. 3 Stripe position (deg.) 1 5 5 1 1 5 5 1 Control Nive 5 s 5 4 3 2 1 1 2 5 4 3 2 1 1 2 Fig. 7. Reltive chnges in wing stroke mplitude response nd closed-loop fixtion ehviour in single flies. () zimuth position of the lck stripe inside the flight simultor (lue, left scle) nd corresponding chnges in the reltive differences etween left nd right stroke mplitude (dw) (red, right scle) of control niml. () Time trces similr to ut of nive fly. Sequences show the flies ehviours 1 12 s fter flight initition. 5 s dw (deg.)
Significnce of experience in flight ehviour 3383.5.4 C.25 E.25 Control single fly.2 Control ll flies.125 Control Nive Normlized FFT mplitude.5.25 2 4 6 8 1 Nive single fly 2.4 D.2 4 6 8 1 Nive ll flies.1 1 2 3 4 5 F Nive minus control 2 4 6 8 1.1 2 4 6 8 1 1 2 3 4 5 Frequency (Hz) Fig. 8. Fst-Fourier trnsformtion (FFT) spectr clculted from the reltive differences etween left nd right stroke mplitude (dw) of 3 min closedloop flight sequence of control nd nive flies. () Non-filtered (grey) nd filtered dt (red) of Fst-Fourier spectrum, clculted for single control fly in nd nive fly in. (C,D) verged FFT spectr derived from 2 control (red) nd 3 nive (lue) nimls. Grey res indicte stndrd devition of the men. (E) FFT trces plotted in C nd D. (F) Difference in FFT spectr etween oth tested groups of flies. To chieve optomotor lnce, the control group closely djusted its men turning rte (519deg.s 1 ) to ren velocity (5deg.s 1 ), which is similr to wht hd een found in previous experiments (Mronz nd Lehmnn, 28). y contrst, nive flies compensted for only pproximtely hlf of the stimulus speed (27deg.s 1 ), lthough their solute turning rte ws similr to the rte of the control group (739 vs 782deg.s 1, P=.46; Tle1). Superficilly, the ltter finding suggests tht nive flies were hndicpped in mintining their flight heding during smooth nd sccdic turns, similr to wht is found in tethered flight. We oserved no difference in men distnce etween the flight pth nd the ren centre mesure tht is thought to reflect the production of centripetl forces during turning flight in circulr ren (Mronz nd Lehmnn, 28). Moreover, none of the tested prmeters ws significntly different in the two groups during the initil tke-off period (.5s; Tle1). In generl, oth groups performed sccdic flight turns in the counter-clockwise direction (ccw) ut lso, less frequent, clockwise (cw), ginst the direction of the rotting ren (Tle 1). Fig. 9I,J shows tht the ngulr velocity profiles were lmost symmetricl round pek turning rte for oth sccde directions ( ms), ut yielded differences etween groups of flies during clockwise sccdic turns (Fig. 9I). Clockwise rottions of the controls were preceded y smll counterturns in the direction of the visul pnorm (lck rrows in Fig.9I), wheres this sutle modultion in velocity ws missing in the nive flies. In contrst to horizontl velocity, pek turning rte within the sccde ws similr under ll conditions ut clockwise totl turning ngle ws significntly lrger in the nive (157deg.) thn in the control group (18deg.; Tle1). DISCUSSION Internl representtion of the visuo-motor system In the present study we investigted the role of experience in the flight ehviour of the fruit fly Drosophil. The tethered nd freeflight dt suggest tht most flight ehviours seem to e geneticlly predetermined ecuse ll nimls exhiited similr men stroke mplitude nd stroke frequency, nd similr open-loop responses to visul stimultion (Figs 4 8). In short free flight outs, pek sccdic turning rte, ngulr ccelertion, pek horizontl speed nd flight ltitude were lso similr in nive nd control flies (Fig. 9, Tle 1). Nevertheless, flight in nive flies without previous flight experience differs from tht of control flies in severl key prmeters. consistent feture of ll tested flies in the simultor ws the position-dependent modultion in yw torque towrds the stripe moving in n open loop (Fig. 4). Thus, even nimls without prior flight experience oviously hve n internl representtion of how the movement of their wings trnslte into rel world ody movements. This visuo-motor gin or response strength is not constnt within the visul field of the niml ut increses when the stripe moves in the frontl ±9 deg. visul field, which corroortes previous results (Fig.5) (Heisenerg nd Wolf, 1984; Reichrdt nd Poggio, 1976). In contrst to the control group, however, nive flies showed inferior flight control when fixting the stripe in open-loop conditions (25% less mplitude difference; Fig. 5) nd performing optomotor ehviour during free-flight conditions (Tle 1), oth indicting flight-dependent dpttion of the visuo-motor gin in the experienced nimls. However, the inferior fixtion in the first open-loop sequence vnished fter flying the nimls under closed-loop conditions, ecuse in the finl openloop sequence the ehviours of nive nd control flies were no longer significntly different. Moreover, nive fruit flies hd less precise yw torque-equivlent mplitude control thn the experienced nimls (Fig. 7). In the following sections we discuss the internl representtion of motor ctions in greter detil, presenting two lterntive hypotheses to explin our results: the neuronl experience hypothesis suggesting tht there re some elements of lerning nd fine-tuning involved during the first flight experiences in Drosophil nd the musculr exercise hypothesis. Owing to the lck of
3384 T. Hesselerg nd F. O. Lehmnn D G α E H C F 1. Normlized trnsfer proility 5 cm Fig. 9. Typicl flight pths of freely flying Drosophil melnogster, trnsfer proility nd flight sccdes for the two experimentl conditions. ( C) Flight pths of three experienced nimls (control) flying in response to the counterclockwise rotting freeflight ren (ngulr velocity, 5 deg. s 1 ). Flight time ws pproximtely 7.9 s in, 4.7 s in, nd 2.9 s in C. (D F) Nive flies flying in similr conditions s in. Flight time is pproximtely 4. s in D, 1.2 s in E, nd 1.3 s in F. Note tht in some cses the fly wlked from the pltform to its strting position (lck dot). The red rrows indicte prominent sccdes. The inner white line represents the dimeter of n inner trnslucent cylinder of the free-flight ren nd the red cross indictes the position of the strting pltform. (G) Trnsfer proility verged over 16 control flies nd (H) trnsfer proility of 13 nive flies. (I) ngulr velocity profile of clockwise (cw) nd (J) counter clockwise (ccw) sccdes in the rotting ren. Grey re indictes time in which totl turning ngle nd ngulr velocity were determined. Red, control flies; lue, nive flies. N=16 (cw, red); 13 (cw, lue); 15 (ccw, red) nd 1 (ccw, lue) sccdes. α,sccdic turning ngle. Turning rte α (1 3 deg. s 1 ). 2 1 I cw 1 1 12 6 6 12 12 6 6 12 Time (ms) 2 1 J ccw experimentl dt, we will not further discuss other hypotheses such s chnges in viscoelstic properties of the thorx nd/or wings or in the concentrtion of neuromodultors ( neuromodultory hypothesis ). Octopmine, for exmple, hs profound effects on the physiology of muscles nd neurons, nd could e elevted in controls s result of pre-experimentl flight ctivity (Chpmn, 1998; rems et l., 27). lthough we cnnot completely exclude this explntion, the hypothesis ssumes tht flight outs t lest 25 3min prior to the experiment ffect the initil open-loop testing. eril performnce nd neuronl experience hypothesis In ddition to the difference in the initil visuo-motor gin ( 2 s flight time) etween the two experimentl groups during open-loop flight (Fig. 5), nive flies lso showed sutle differences in closedloop flight when flown under tethered- (2 2 s) nd free-flight conditions (Tle 1). Most notly, nive flies showed higher degree of ctive steering during fixtion of the stripe when tethered thn the controls, which resulted in significnt higher solute difference of wing stroke mplitude etween the two wings. Nive flies my potentilly compenste for this reduction in fine control of their motor ctions (stroke mplitude) y incresing the temporl rte of steering mnoeuvres. The distortion of the FFT spectrum shown in Fig. 8E might indicte such process. The ltter ehviour prtly restores the reduction in turning efficiency for yw control nd thus flight heding stility. This result my suggest tht the chnges in yw control nd thus the chnges in fixtion ehviour result from n ctive ehviourl process supporting the neuronl experience hypothesis, rther thn from reduction in the ility of the nive nimls to mximise their stroke mplitudes. lthough Fig. 5 clerly shows tht the difference in left-minusright stroke mplitude quickly ceses with flight time, the underlying sensu-motor mechnism for this decy is less cler. There re t lest two possile explntions for this finding. First, the response difference vnishes ecuse the nive nimls experience closedloop feedck conditions etween oth open-loop sequences, llowing the flies to djust their neuromusculr system similr to tht of control nimls. This would require short-term djustments vi physiologicl chnges in neurl ctivity within 3min of closedloop flight. In this scenrio, we would lso expect tht the response difference etween nive nd control flies persists when repetitively testing the nimls under open-loop conditions, without the ppliction of closed-loop feedck conditions. Second, n lterntive explntion would e tht the initilly scored response difference ( 2 s; Fig. 5) decreses with incresing flight time, independently of the experience collected during the closed-loop feedck flight sequence. Experiments plnned in the future should
Significnce of experience in flight ehviour 3385 Tle 1. Free-flight chrcteristics of experienced (control) nd nive fruit flies fter tke-off in free-flight ren rotting counter clockwise t 5 deg. s 1 Prmeter Control Nive d.f. P Numer of flies 16 13 Flight time (s) 2.38±1.73 (.5) 1.81±1.6 (.5) 27.31 Distnce to inner cylinder (mm) 47.1±7.7 (46.8±11.8) 41.6±15.2 (42.4±8.88) 27.18 (.14) Flight ltitude (mm) 34.2±16.2 (2.7±14.9) 32.9±13.5 (13.1±6.66) 27.82 (.11) Horizontl speed (m s 1 ).43±.8 (.34±.9).33±.7 (.31±.9) 27 <.1** (.33) Pek horizontl speed (m s 1 ).84±.17.73±.17 27.8 Horizontl ccelertion (m s 2 ) 1.14±.28 (4.79±2.12) 3.8±1.41 (4.9±1.4) 27 <.1*** (.31) Turning rte (deg. s 1 ) 519±159 (22±372) 27±242 (169±635) 27.3** (.86) solute turning rte (deg. s 1 ) 782±137 (83±231) 739±174 (84±317) 27.46 (.72) ngulr ccelertion (1 3 deg. s 2 ) 24.±7.28 (29.3±11.9) 24.3±6.99 (28.9±14.) 27.93 (.94) Totl numer of sccdes cw 17 2 Totl numer of sccdes ccw 134 55 Sccdic turning ngle cw (deg.) 18±12 157±11 35 <.1* Sccdic turning ngle ccw (deg.) 12±3. 113±5. 187.18 Pek turning rte cw (deg. s 1 ) 1557±448 1697±418 35.44 Pek turning rte ccw (deg. s 1 ) 1533±142 1567±472 187.79 Pek horizontl speed is the 5% uppermost vlues in the flight sequence. Horizontl nd ngulr ccelertions were ech clculted from solute velocity estimtes. Vlues re mens ± stndrd devition; vlues in prentheses re mens of the first.5 s fter flight initition. Smple size for sccde sttistics ws the totl numer of sccdes derived from ll flight sequences. cw, clockwise rottion of the fly (negtive turning rte); ccw, counter clockwise rottion (positive turning rte). P-vlues were from two-tiled independent t-test on dt mens; *5%, **2% nd ***1% significnce level. llow us to distinguish etween these long- nd short-term djustments, nd thus to possily clrify the nture of the mesured chnges. The ove conclusion is lso supported y two further findings. First, freely flying nive flies hd more errtic flight pth due to n pprent loss of precise heding control nd showed insufficient optomotor response during free flight in the rotting ren, lthough they flew t the sme men ltitude nd thus lift production (Tle1). Second, nive nimls exhiited chnges in reltive frequency nd velocity profile of clockwise nd counter-clockwise free-flight sccdes, respectively. Experienced flies, for exmple, elicited pproximtely 9% of the sccdes in the counter clockwise direction nd thus in the direction of the rotting ren. y contrst, nive flies more often turned clockwise (73% counter clockwise rottions), contrry to wht would e expected from optomotor stimultion, nd lso yielded higher vrinces in their responses. possile explntion for the oserved chnges is tht flight sccdes in nive flies re more often triggered y retinl slip due to imge expnsion or contrction on the lterl eye regions, rising from more frequent pproches towrds the visul pnorm (Duistermrs et l., 27). Musculr exercise hypothesis The finding tht in the entire free-flight sequence, men horizontl speed in nive flies is pproximtely 23% less thn in the control group (Tle 1), my support the musculr exercise hypothesis mentioned efore. This theory suggests tht the weker response in nive flies results from either decrese in mechnicl power output of the indirect flight muscle (IFM) or y physiologicl chnges in flight control muscles cused y lck of exercise, rther thn from lck of previous experience. lthough our free-flight dt re consistent with oth the neuronl experience nd the musculr exercise hypotheses, there is little well-founded evidence for the ltter for the following resons. First, previous studies on flight ctivity in house nd fruit flies hve repetedly shown tht n increse in musculr exercise cuses n increse in mortlity rte s result of incresed oxidtive dmge (Mgwere et l., 26; Yn nd Sohl, 2). Consequently, it is less likely tht exercise improves muscle performnce of the IFM. Second, lthough men horizontl speed ppered to e weker in nive flies thn in the controls, pek performnce ws nerly identicl in oth groups nd men ody ccelertion ws even pproximtely three times higher in the nive flies (Tle 1). Third, under tethered flight conditions, we otined slightly higher mesures for pek erodynmic force production in the controls thn in the nive nimls, superficilly supporting the muscle exercise hypothesis (Fig. 6). However, sttisticl tests did not yield ny sttisticlly significnt differences etween the two mesures. Collectively, our results indicte tht nive nimls pper to e cple of producing similr mximum musculr mechnicl power output y the IFM nd control muscles s the controls, which implies, gin, tht their flight ehviour ws shped y the lck of previous experience nd not y power ttenution. Conclusions Conventionlly, it is not elieved tht invertertes such s insects re cple of showing motor lerning sed on sensory input or from experience, s hs een found in vertertes (Yod et l., 24). Undoutedly, most flight ehviour in Drosophil seems to e geneticlly predetermined, ecuse nive flies show seemingly norml flight ehviour when rndomly relesed into the lortory. However, since relevnt studies hve repetedly emphsised the evidence of ehviourl plsticity nd lerning in insects, we should revise our simplistic view on insects s geneticlly determined flight mchines. More recently, proof of complex motor lerning in insects hs een incresing nd includes the lerning of specific motor sequences in nts tht hd to nvigte through mze (Mcqurt et l., 28), the ility of fruit flies to ssocite visul cues with specific set of motor commnds [yw torque (Wng et l., 23)], nd pilot experiments on the updte of long-lsting djustments of the visuo-motor gin in wild-type nd lerning mutnts of Drosophil, trined t vrious feedck conditions during tethered flight (T.H. nd F.O.L., unpulished results). In generl, dt on the cquisition of everydy complex motor skills from previous experience re rre in insects. Our dt on the plsticity of the effect of motor commnds on ody movements in Drosophil thus helps to understnd the concepts of motor lerning in model systems for flight. lthough flying vertertes pprently rely more strongly on the dvntge of motor lerning to improve
3386 T. Hesselerg nd F. O. Lehmnn their flight efficiency, gility nd mnoeuvrility even smll djustments in control properties of the feedck cscde in insects my dvnce their fitness, for exmple, during escpe from predtors, mting flights or eril comts. Therefore, studies on the plsticity of motor skills re lso of interest for engineers who im to design the future genertion of micro-ircrfts sed on flpping wing propulsion. We would like to thnk Nicole Heymnn nd Meliss Sonnenmoser for their help with the experiments nd Ursul Seifert for criticlly reding the mnuscript. This work ws funded y the ionics grnt 313772 of the Germn Federl Ministry for Eduction nd Reserch (MF) to F.O.L. REFERENCES utrum, H. (1958). Electrophysiologicl nlysis of the visul system in insects. Exp. Cell Res. Suppl. 14, 426-439. ender, J.. nd Dickinson, M. H. (26). comprison of visul nd hlteremedited feedck in the control of ody sccdes in Drosophil melnogster. J. Exp. iol. 29, 4597-466. lesing,. nd Cruse, H. (24). Stick insect locomotion in complex environment: climing over lrge gps. J. Exp. iol. 27, 1273-1286. orst,. (199). How do flies lnd? From ehvior to neuronl circuits. ioscience 4, 292-299. rems,., Christinsen, F., Pflüger, H. J. nd Duch, C. (27). Flight initition nd mintennce deficits in flies with geneticlly ltered iogenic mine levels. J. Neurosci. 27, 11122-11131. Chpmn, R. F. (1998). The Insects. Cmridge, M: Hrvrd University Press. Dvid, C. T. (1978). The reltionship etween ody ngle nd flight speed in freeflying Drosophil. Physiol. Entomol. 3, 191-195. Duistermrs,. J., Chow, D. M., Condro, M. nd Frye, M.. (27). The sptil, temporl nd contrst properties of expnsion nd rottion flight optomotor responses in Drosophil. J. Exp. iol. 21, 3218-3227. Ellington, C. P. (1984). The erodynmics of insect flight. VI. Lift nd power requirements. Philos. Trns. R. Soc. Lond. iol. Sci. 35, 145-181. Fry, S. N., Symn, R. nd Dickinson, M. H. (23). The erodynmics of free flight mneuvers in Drosophil. Science 3, 495-498. Frye, M.. (27). ehviorl neuroiology: virting gyroscope controls fly steering mneuvers. Curr. iol. 17, 134-136. Gee, C. E. nd Roertson, R. M. (1996). Recovery of the flight system following ltion of the tegule in immture dult locusts. J. Exp. iol. 199, 1395-143. Grohmnn, J. (1939). Modifiktion oder Funktionsreifung? Z. Tierpsychol. 2, 132-144. Hmer, K. C., Schreier, E.. nd urger, J. (22). reeding iology, life histories, nd life history-environment interctions in seirds. In iology of Mrine irds (ed. E.. Schreier nd J. urger), pp. 217-261. oc Rton, FL: CRC Press. Heisenerg, M. nd Wolf, R. (1984). Vision in Drosophil. erlin: Springer-Verlg. Hesselerg, T. nd Lehmnn, F. O. (27). Turning ehviour depends on frictionl dmping in the fruit fly Drosophil. J. Exp. iol. 21, 4319-4334. Jckson, R. R., Crter, C. M. nd Trsitno, M. S. (21). Tril-nd-error solving of confinement prolem y jumping spider, Porti fimrit. ehviour 138, 1215-1234. Lgris, J. C., Reeds, J.., Wright, M. H. nd Wright, P. E. (1998). Convergence properties of the nelder-med simplex method in low dimensions. SIM J. Optimiztion 9, 112-147. Lehmnn, F. O. nd Dickinson, M. H. (1997). The chnges in power requirements nd muscle efficiency during elevted force productions in the fruit fly Drosophil melnogster. J. Exp. iol. 2, 1133-1143. Lehmnn, F. O. nd Dickinson, M. H. (1998). The control of wing kinemtics nd flight forces in fruit flies (Drosophil spp.). J. Exp. iol. 21, 385-41. Lehrmnn, D. S. (1953). critique of Konrd Lorenz s theory of instinctive ehvior. Q. Rev. iol. 28, 337-363. Lorenz, K. (1937). Üer die ildung des Instinktegriffes. Nturwissenschften 25, 289-331. Mcqurt, D., Ltil, G. nd eugnon, G. (28). Sensorimotor sequence lerning in the nt Gigntiops destructor. nim. ehv. 75, 1693-171. Mgwere, T., Pmplon, R., Miw, S., Mrtinez-Diz, P., Portero-Otin, M., rnd, M. D. nd Prtridge, L. (26). Flight ctivity, mortlity rtes, nd lipoxidtive dmge in Drosophil. J. Gerontol. iol. Sci. Med. Sci. 61, 136-145. Mtheson, T. (1997). Hindleg trgeting during scrtching in the locust. J. Exp. iol. 2, 93-1. Myer, M., Vogtmnn, K., usenwein,., Wolf, R. nd Heisenerg, M. (1988). Flight control during free yw turns in Drosophil melnogster. J. Comp. Physiol. 163, 389-399. Möhl,. (1988). Short-term lerning during flight control in Locust migrtori. J. Comp. Physiol. 163, 83-812. Mronz, M. nd Lehmnn, F. O. (28). The free flight response of Drosophil to motion of the visul environment. J. Exp. iol. 211, 226-245. Petersen,., Lundgren, L. nd Wilson, I. (1956). The development of flight cpcity in utterfly. ehviour 1, 324-339. Pick, S. nd Struss, R. (25). Gol-driven ehviorl dpttions in gp-climing Drosophil. Curr. iol. 15, 1473-1478. Reichrdt, W. nd Poggio, T. (1976). Visul control of orienttion ehvior in the fly Prt I. quntittive nlysis. Q. Rev. iophys. 9, 311-375. Tmmero, L. F. nd Dickinson, M. H. (22). Collision-voidnce nd lnding responses re medited y seprte pthwys in the fruit fly, Drosophil melnogster. J. Exp. iol. 25, 2785-2798. Tmmero, L. F. nd Dickinson, M. H. (22). The influence of visul lndscpe on the free flight ehviour of the fruit fly Drosophil melnogster. J. Exp. iol. 25, 327-343. Tinergen, N. (1951). The Study of Instinct. Oxford: The Clrendon Press. Venner, S., Psquet,. nd Leorgne, R. (2). We-uilding ehvior in the orweving spider Zygiell x-nott: influence of experience. nim. ehv. 59, 63-611. Wgner, H. (1986). Flight performnce nd visul control of flight of the free-flying house fly (Musc domestic L.). II. Pursuit of trgets. Philos. Trns. R. Soc. Lond iol. Sci. 312, 553-579. Wng, S., Li, Y., Feng, C. nd Guo,. (23). Dissocition of visul ssocitive nd motor lerning in Drosophil t the flight simultor. ehv. Processes 64, 57-7. Wilson, D. M. (1961). The centrl nervous control of flight in locust. J. Exp. iol. 38, 471-49. Witt, P. N., Rwlings, J. O. nd Reed, C. F. (1972). Ontogeny of we-uilding ehvior in two or-weving spiders. m. Zool. 12, 445-454. Yn, L. J. nd Sohl, R. S. (2). Prevention of flight ctivity prolongs the life spn of the housefly, Musc domestic, nd ttenutes the ge-ssocited oxidtive dmge to specific mitochondril proteins. Free Rdic. iol. Med. 29, 1143-115. Yod, K., Kohno, H. nd Nito, Y. (24). Development of flight performnce in the rown ooy. Proc. iol. Sci. 271, 24-242.