of Attraction of horticola (Coleoptera,

Attraction of Phyllopertha horticola (Coleoptera, Scarabaeidae) males to fragrance components of Ophrys flowers (Orchidaceae, section Fuciflorae)

ANNA.KARIN BORG-KARLSON

Borg-Karlson, A.-K.: Attraction of Phy llopertha horticola (Coleoptera, Scarabaeidae) males io fragrance components of Ophrys flowers (Orchidaceae .section Fuciflorae). ITriidgrirdsbor- rens hanar (Phyllopertha horticola) attraheras till komponenter i blomdoften frintre Ophrys arter (Orchidaceae . sektion Fuciflorae).1 - Ent. Tidskr. t09: 105-109. Umei, Sweden 1989.

ISSN 001-l-886x.

The North-European scarabaeid beetle Phyllopertha horicola has been obscrvcd to visit and exhibit pseudocopulatory behaviour on the nectarless flowers of C)pftrys species (section Fuci /orae). To evaluate the role of chemical signals in the beetles' attraction response io the flow- ers and to determine the active compounds involved, males from a swarming Population on Oland (Sweden) were tested for their attraction to different odour samples: hexane exlracts of Ophrys flower labella, hexane extracts of male and female beetles, and different blends of compounds identified in Opftrys flowcr fragrances. Male beetles were most strongly attracted to a blend of short-chained aliphatic alcohols present in extracts of O. hoktcericea labella.

A.-K. Borg-Karkon, Ecological Research Sration of IJppsala Ilniversity, Olands Skogshy 6280, 5-386 00 Fiirjestaden, Sweden and Departmen of Orgqnic Chemistri', The Royal Institute oI 'feclnology. S-100 41, Stockhobn, Sweden.

lntroduclion

The scarabaeid bcetle Phyllopertha horticola (L.)

has repeatedly been observed to visit flowers of Ophrys L., sectioll Fuciflorae Rchb.f. (Kullen- berg 1973, Warncke & Kullenberg 1984. Borg- Karlson 1987). The often white or cerise sepals of Oplrrys flowers are similar in coloration to ^th€

beetles' food plants Cisrr.r (Cistaceae) and Crata- egus (Rosaceae). It has been suggested that these colours may act as visual cues encouraging both male and female beetles to visit and forage on Ophr,r,s flowcrs and thcre bv incidentally pollinate them ^(Ferlan in lit. to Kullenberg. Kullenbcrg 1961). However, recent observations (Forster, Paulus, in lit. to Kullenberg 7976,7982,1983) re- vealed that P. horticola malesbehave as ifsexually stimulated when visiting O. holocericea (Btrm.

fil.) Greuter l: ^O. fuciflora ^{(Cranz) Moenckl} flowers. Other scarabaeid beetles, such as Tropi- nota squalida Scop. and Oxythyrea funesta Poda, also visit and pollinate various Opftrys species in the section Fuciflorae (Ktsllenberg 1961).

It has not been established whether the beetles

arc attracted/excited primarily by odour, or whethcr visual and tactile cues are also necessary to elicit the complete sequenced behavioural re- sponses leading to pollination by pseudocopula- tion.

The purpose of this study was to experimentally examine, using behavioural tests, the role ofolfac- tory stimuli in the pollination relationship betwe- en the beetle Phyllopertha horticola and the spe- cies Ophrys holocericea, O. scolopax Cav. ^and O.

t

ent h re d i nif e r a ^tN illd.

Materials and methods

The study took place on Oland, one of the large is- lands in the Baltic Sea closc to the mainland of southcrn Sweden. Odour attraction tcsts were performed from end May to mid Junc 1983-85 on a field population of beetles inhabiting the border of a grass-lined gravel road surrounded by hazel- bushes (Corylus avellana L.).

To test the beetles attraction to ^different

106 Anns- Karitt l)org-Korlson

h i.+o 66p.

Fig. l. The odour-guidcd rcsponses of Pryllopertha hor- ricola male beetles are divided into four behaviour tvpcs based on increasing degrees of attraction/excitation: ⁽ l) p : uninlluenced passage, (2) q.i. : quick inspection, (3) h. i. : hovering inspection and w.i. = ^{walking inspec-} tion, ⁽¹⁾ h.i.-a.cop. : hovering inspection followed by

"copulation attempt" or w,i. ^q,cop. : walking inspec- tion folkrwed bv "attempted copulation", The insects are regar(lcd as strr',nglv attracted in the behaviour types 3 and 4.

Det dottinducerade beteendet som trddgirdsborrehanar utfdr vid attrappen Ar uppdelat i fyra olika typer med sti- gandc attraktivitctsgrad: (l) 2 : ^{passage, (2)} S.i. ⁼ snabb inspektion, (.3) n.i. = sidsvingande eller r,r,.i. = krvpandc inspcktittn. (1) h.i.--a.cop. eller w.i.-a.cop. = sidsvlngandc cllcr krypande inspektion som slutar med fiirsitk till kopulation pe artrappen. Insekterna iir starkt attraherade om de reagerar med beteendetyp 3 cllcr 4.

odours, eight different samples were presented to heetles within the confines of the swarm. Extracts were studied of Ophrys scolopax and O. lenth- rediniferu labella, and of female and inale /'ftyl- lopertha horticola bodies. Fresh Oplrrys labella were extracted in redistilled hexane (5 labclla/ml hexane, Merck) for three days at 4'C and l-2 days old P. hortitolu bcctles were extracted in redistil- led pe ntanc (2 whole bodies/ml pentane, Merck) for 24 hours at 20'C.

The following three authentic blends of com- pounds identified in scents of Opftrys flowers were studied (volumes in pl; each blend diluted in ^1.9

m.l hexane): Alcohol blend: l.hexanol 2, cis-3-

hexcn-l-ol 2. 1-octanol 10, l-hexadecanol l0;

Acid blend: pcntanoic acid 2, hexanoic acid 4, hcptanoic acid 10, octanoic acid 2, nonanoic acid 20; Ophrys tenthredinifera extruct blendi l-hexa- nol 2, ci.r-3-hexen- l -ol 2, 1-octanol 10, l-hexade- canol 10, 6-methyl-5-hepten-2-one 6. pentanoic acid 2, hexanoic acid 4. heptanoic acid l(). oc-

tanoic acid 2. nonanoic acid 20. citral4, geraniol 1,

citronellol 4. geranylacetone 4.

The compositions of the floral blends were bascd on GC-MS (gas-chromatography mass spectrometry) analyses madc earlicr (Kullenberg ct al. 1984, Borg-Karlson 1987).

The lest samples were applicd to dummies-

picces of black velvet (5 x l0 mm) placed on insect pins and covercd with green tullc. Abovc the tulle was attached an odourlcss piece of velvet to in- crease the visual and tactile stimuli of the dummy.

Samples were tested one bv one in random order, for 5 minutes each, and in +10 repllcates. Re- sponses of beetles, within 0.2 m from the dummv, were noted. Results were evaluated statistically using the chi-square test for 2 x 2 contingency ta- blcs. For each test sample, the sum of behaviour types 3 and 4 [hovering (h.i. ), walking-inspection

(w.i.), and alighting rcsponscs on the dummy (h.i.-a.cop., w.i.-a.cop.)], i.c. thc ^{strongly at-}

tracted beetles (Fig. l), was dividcd by the total number of responses (N). Portions of thc be- havioural sequences were filmed using a Paillard Bolex 16 mm film camera.

Voucher beetle specimens are deposited at the Ecological Research Station of Uppsala Univer- sity on Oland. Taxonomy and nomenclature of Ophrys is based on Nelson (1962), sce also Borg- Karlson ( 1987) and rcfcrcnces therein.

Results

In the mornings, male heetles started to walk whcn ambient temperaturc rcachcd 13"C (at 1.5 m above ground) and to swarm at lzl-15"C. Swarm- ing usually lasted for about three hours. Female beetles were never seen swarming, bui were fre- quently observed feeding on leaves and ^grasses in the vicinity.

Only males were attracted to the dummies. The bchavioural rcsponses of malc bectles. both flying and walking, to the dummies werc classified into 4 typcs as follows, listed in ordcr of increasing at- traction/excitation (Fig. 1).

1. p = uninfluenced passage: by-passing without de- creasing flight speed or changing flight direction.

2. q.i. = ^quick inspection: flight direction changed to- wards dummy, sometimcs rcsulting in touch.

3. r.i. : hovering inspection: mcandcring flight towards

the odour source. hovering in front of it, sometimes fly-

ing around it, always facing tho dummy. w.i. = walking

inspection: walking on ground (x in vcgetation towards

clumml but without touch.

Attractiort oJ Phyllopertha nales to Ophrys flowers ¹⁰⁷

Tab. I . Respon sc s

tf P htlloperrha hortico,/a males towards each of the eight samples tested; f( 3 + 4) = the proportion of attracted beetles with behaviour iype 3 and 4 (e.g., h. i., w.i., h.i.-a.cop., w.i.-a.cop.) in relation to all beetles (within 0.2 m of the test dummy) for a given sample; f(4) = ^the proportion of attracted beetles with behaviour type ,[l N = ^thc total number of beetles observed for a given _test sample; n = the number of replicates for each sample.

Trddgirdsborrens betqcndcsvar gentemot 8 testamnen: f(3+4) : proportionen attraherade hanar med beteendetyP -j eller 4, jiimfort med alla hanar inom 0.2 m fran rest-attrappen; f(4) = proportionen attraherade hanar med beteende tr"p,+: N = totala antalet observerade skalbaggar; n = ^antal testtillfiillen frir varje iimne.

Sample f(3+4) (4)

Alcohol blend

O p hrys

o lo pax labella extract Phyllopertha horticold nrale body extract P, horticolalemale body cxtract O. tenthredinifera extract blcnd O. tenthre dinifero labclla extract Acid blend

Hexane blank

0.36*

0.25- 0.14 0.13.*

0.12 0.08 0.06 0.05

0.19 - 0.10 0.09 0.06 0.10 0.00 0.02 0.02

10 8

l0

8

l0

4 9 10 64

40 58 70 50 25 54 56

' 'iSnrlicant!.l difl.r.nr lronr tllt hlanl lX:. p<lr Ill5) '- \ignrfrcanlh difterenr tnnn rhc Allohrn bbnd (X:. p<0.05)

4. h .i.--a. cop. - hovering inspection followed by descent and walking on dummy.'fhe beetle performs move- ments that appear to represent a copulation attempt, tt.i.-a.cop. : walking inspection followed by contact with the durnmv and seeminglv attempted copulation.

Extracts of O. scolopax labella, which are domi

naled by the alcohols l-hexanol and 3-hexen-1-ol (Borg-Karlson 1987), proved to be highly attrac- tive to P. horticola malcs (Tab. 1). The strongest attraction, howcvcr, was eliciled by the alcohol blend. This blend differed significantly in attrac-

tivity from the P. horticola female extract (Xr, p<0.05), the acid blend and the hexane blank ^(X'?,

p<0.01), which released the two ^{weakest re-}

sponses. Attraction to the extract blend of O.

tenthredinifera, which contained constituents of

both the alcohol and acid blends. was much lower than that 1o O. sutktpux cxtracts, but similar to those observed for hody cxtracts of male and of female P. horticola. When a highly excited male beetle responds to the alcohol blend the abdomen is stretched out, the copulation apparatus fully clongated, and thc bcctle is eagerly grasping the piece of velvct. This bchaviour must be inter- preted as attempted copulation, and has not becn observed when the beetle is feeding on its food plants (Kullenberg pers. comm.). The very few approaches displayed by beetles towards the con-

trol hexane dummy suggest that the ^visual

stimulus of the velvet might be negligible com- pared to the olfactory stimuli of the tested odour samples in eliciting copulation.

Discussion

Rcsults of the present odour-attraction Iests indi- cate that P. horticolq malcs are attracted to vol- atile compounds prescnt in flower fragrances of the studied Op&rys species. White and cerise col- our cues were excluded in the experiments and the beetles seemed to be only slightly, if at all, af- fected by the visual stimulus of the dummy on which odours were applicd. Only males were at- racted to the test-samplc dummies and, when highly excited, they performcd movements that

may be interpreted as "pseudocopulation".

Aliphatic alcohols seem to be of greatest impor- tance in releasing this behaviour.

The body extract of female beetles was only weakly attractive to males which is in contrast to the results from a recent study of P- ^diversa Watcrhousc where the female body extract was

vcry attractive to conspecific malcs. This may, howcver, be influenced in part by the age of the fenrales. The dilution of the P. diversa female ex-

tract was found to be of minor importance (Kawasaki & Tamaki 1985).

Thc aliphatic alcohols tcsted are not restricted in occurrence to the three Opftrys species exam- ined here. but are also found in large amounts in the fragrance emitted from O. lutea Cav. and O.

.ftrsca Link (Borg-Karlson et al. ^{1985, Borg-}

Karlson & Tengd 1986). However, to my knowl- cdge thcre are no observations of P. horticolavis-

iting these Oplzry.r specics, which may be

lO8 Anna- Karin Borg- Karlson

explained by differences in the blooming times and habitats of diffcrent plant species and in swarming timcs ofdiffercnt beetle populations. P.

horticola belongs to a family that are known to re- spond to a variety of compounds (Nilsson A. pers.

comm.). l-Hexanol and cis-3-hexen- 1-ot, both present in the alcohol blend, are common in plants and probably function as food attractants to many insects. However. an odour induced sexual attrac-

tion to the aliphatic alcohol blend and to the Ophrys lab'ella extracts is strongly suggested by three observations: (l) beetles oriented towards the odourife rous dummy in the al\sence of white or cerise colours (visual stimuli), (2) only males were attracted to thc dummies. (3) the behaviour

of the descending nrales on the odouriferous dummv resembled the initiation of a copulation.

This behaviour was not initiated by the hexane dummy (tactile stimuli not enough).

It is interesring to note that the pollination of Ophrys orchids in certain habitats are performcd by insects other than bees. The importance of bee- tles as pollen vectors and their flower preference patterns in thc genus Ophrys, are not known.

Their flower visits are most likely of low specific-

ity. leading to interspecific pollen transfer and mav explain some of the hybrids found. The regu- lar pollen vectors in the Opftrys section Fuciflorae are known to be males of the solitary bees Eucera and Terralonia (Kullenberg et al. 1984a,b).

Acknowledgements: I thank Prof. Bertil Kullenberg for his enthusiastic support and valuable discussions con- cerning this study. and tbr his excellent filmings of beetle behavioural sequences. I also want to thank Mrs Barbro Holmlund for skilful technical assistance and Dr Anders Nilsson (Uppsala) and Dr Lcnnart Agren for valuable discussions and Dr Heidi Dobson for linguistic revision of the manuscript. Financial support from the Swedish Natural Science Research Council. the Bank of Swedcn Tercentenarv Foundation. and the Axel and Margarct Ax:son Johnson Fou ndation is gratefully acknowledged.

References

Borg-Karlson. A.-K., lJergstrdm, G., & Groth. L l98-5.

Chemical basis for the relationship between Ophrys orchids and their pollinators I: Volatile compounds of O. lutea and O. fusca as insect mimetic aftractants/

excitants.

- ^{Chem. Scr.} 25:283-294.

Borg-Karlson, A.-K. & Teng<i. J. ^{1986. Chemical}

mimetism'l Key substances in the Ophrys lutea -

ADdrena pollination relationship.

- ^J. Chem. Ecol.

12: 1927-1941.

Borg-Karlson. A.-K.. l9ll7. Chemical basis for the re- lationship between Ophrys orchids and their pol- linators III: Volatile compounds of species in the

Ophrys sections Fuciflorae and Bombyliflorae as in- sect mimetic altractants/ex.itanls.

-Chem. ^{Scr. 27:}

313-325.

Kawasaki, K. & Tamaki. Y. l9tl5. Evidence of the sex

attractant pheromone and activit! rhylhm in Phyl-

lopertha diversa Waterhouse (Coleoptera, Scarabaeidae).

- Appl. Ent. Zool. 20 (2): 137 ^142.

Kullcnberg. B. t961, Studics in Ophrys pollination.

Zool. Bidr. Uppsala -j4: l--j.10. .51 plates. -

Kullenberg. B. 1973. Ncw ohscrvations on the pollina- tion of Ophrys orchids [-. (Orchidaceae).

- ^Zoon.

Suppl. l:9-11.

Kullenberg, B., Borg-Karlson. A.-K. & Kullenberg, A.-L. 1984a. Field studies on the behaviour of the Eucera nigrilabris male in the odour flow from flower Iabellum extract of Ophrys tenthredinitera.

- ^Nova

Acta Reg. Soc. Sci. Upsalicnsis. Ser. V:C,3r 79 l l0.

Kullenberg. 8., Btiel. H., & Tkalci. B. 1984b. Uber- sicht von Beobachtungcn iiber Besuche von Eucera und Tetralonia-Minnchen auf Ophrys-Bliiten (Or- chidaceae).

- ^{Nova Acta} Reg. Soc. Sci. Upsaliensis.

Ser. V:C- 3: 27-.1U.

Nelson, E. 1962. Gestaltwandel und Artbildung erdnen am Beispiel der Orchidaceen Europas und der Mit- telmeerlander insbesondere der Gattung Ophrys.

250 pp. 56 pl. 8 cartes. Chernex et Montreux.

Warnckc. K. & Kullenbcrg. B. ^19x4. Uher.icht vt'n Beobachtungen Besuche von Andrena- und Colletes cunicularius

- Mannchcn auf Ophrys BlLiten (Or- chidaceae).

-Nova Acta Reg. Soc. Sci. Upsaliensis, Ser. V:C. 4l-55

Sammonfattning

Ophrys orkidd.er pollineras i huvudsak av solitdra

bi-, ^grdv- och dolkstckelhanar (Hymenoptera:

Andrenidae, Anthophoridac, Megachilidae, Sphecidae och Scoliidae). Under senare ir har dven minga observationer av besokande och pol- linerande skalbaggar av familjen Scarabaeidae rapporterats inom Ophrys sektionen Fuciflorae.

Dessa orkid6ers vita eller rddaktiga kalkblad har antagils ha stor betydelse fdr insekternas oriente- ring till blommorna. Skalbaggens beteende pi den finludna blomliippen har heskrivits bide som ndringssrik och forsrik till kopulation.

For att utvardera kemiska signalers betydelse fdr skalbaggarnas orientering mot Oprrys-blom- morna,

-gjordes beteende-experiment i falt ^pa

Oland. Atta olika extrakt och substansblandning- ar testades pe triidgardsborrar (Phyllopertha hor- llcola) vilka har observerats som pollinerare pi O.

holocericea (: O. fuciflora). Insekternas reak- tioner infor doftattrappen graderades i fyra be- teendekategorier med stigande attraktivitet vilka scdan anvdndes fdr att beddma attraktionsgraden av olika testemnen.

Endast hanar attraherades, starkast till ^en

blandning bestiende av cis-3-hexen- I -ol, 1-hexa-

nol, 1-octanol och l-hexadecanol (emnen som ocksi finns i blomdoften hos O. holocericea och O. scolopax\. men inte till attrappen med enbart lcisningsmedel. Beteendet pA attrappen med al- koholblandningen kan tolkas som sexuellt upp- hetsat di kopulationsapparaten var helt utdragen

Attraction ol Pht'llopertha males to Ophry"s Jlon,ers ¹⁰⁹

hos vissa hdgexciterade hanar. Tridgirdsborrens hanar attraheras utan visuella stimuli av flyktiga cioftImnen som utsondras frin Opftrys-labellen.

Dcssa attraktiva dmnen finns hos flcra Ophrys- taxa. vilket mojligen kan orsaka korspollination och delvis forklara uppkomstcn av vissa hybrider.

Recensioner

Gdrdcnlors. U., Hall. R.. Hansson. C. & Wilan- dcr. P. 1988. Svensku smitkryp. En bestiimnings- bok till ry'ggnulslosu djur utofti insekler. Student- litteratur. Lund. 147 s. ISBN 9l-44-27301-0. Pris

125 SKr.

Bra bestamningslitteratur pi svenska iir nigot vi entomologer inte air hortskamda med. Svenska smikryp ir diirmed ett mycket viilkommet bidrag fOr oss som ir intresserade av den ldgre faunan.

Till skillnad frin de flesta populdra civersikter har fdrfattarna valt att ge bestiimningsnvcklar till

samtliga familjer. och inte bara presentera ett ax- plock vanliga arter. Pa detta satt luras man inte till att driva sin bcsttimning langre an till vad boken egentligen zir avscdd for. Frir den vetgirige kan detta i sig vara frustrerande. men di far man leta vidare i nigot av de minga verk som refereras till

efter varje nycke l.

Som titeln anger behandlas ej insekterna. men alla dvriga svenska evertebrater. Som regel har man valt att behandla flertalet artrika gruppcr med smivdxla former mer dversiktligt. Detta giil- ler bl a kvalstrcn och de liigre kriiftdjuren, till vil- ka inga familjcnvcklar ges. En introduktions- nyckel leder vidarc till separata nycklar for olika phyla. vilka sedan 6ven de ofta indelas i under- grupper med separata nvcklar. Frir varje familj ges uppgifter om artantal och utbredning i Sveri-

gc. storlek. levnadsmiljoer och vanligen n6gra morfologiska kennetecken utdver de som finns i

n vck larn a.

Nycklarna iir rikligt illustrcrade med teckningar av djur i helfigur eller av detaljer. Det iir genom- giende hog kvalit6 pA teckningarna, som dr till

mycket stor hjelp.

Boken kommer sekert att anvdndas flitigt inom undervisning. I fdrordet nAmns aven att den byg- ger pi kompendicr som anvants pi kurser vid Lunds universitet. Siikert kommer den avcn att

gliidja minga allment naturin tresse rade. bide som hjiitp att identifiera djur och som informa- tionskalla.

En sannolik kiilla till problem for anvAndare ir

att i ^nycklarna blandas morfologiska karaktdrer med sidana som avser levnadssatt och -miljd. Det scnarc kan ofta vara svart att avgora ntr man v61

har djuret isin hand. Extra svirt blir dct niir djur frin 0vergingsmiljOer som t ex brackvatten skall unclcrscikas. Hur viiljer man di mcllan marina el- ler limniska former'? Det kan dven vara svert att tilliimpa de kcinsbundna karaktdrcr som anvzinds.

t ex for vissa av de hogre kraftdjuren, nar det inte anges hur man skrljer k<inen it.

Trots dessa mindre anmirkningar fungerar sf,- kert nycklarna bra. och det ar bara att lyckdnska fdrfattarna till ^ett fint resultat. Boken lr mig ve- tcrligt den enda i sitt slag pi den svenska markna- clcn och dirmed mycket viilbchovlig.

Anders Nilsson

Baccetti, B. (Ed.) 198"7. Evolutionar-v Biology of

Orthopteroid lnsects. Ellis Horwood Series in En- tomology' and Acridology. Chichester. ISBN 0- 7458-0208-7. Pris I190 DKr.

Initiativct till ^denna samlingsvolym togs redan 1960 under den lnternationella Entomologkon- gressen i ^{Wien. Det} visade sig att det fanns ett stort behov av att samla forskare som arbetade mcd systematik och fylogenihos Orthoptera ser?su

/a/.' trll ctt eget mcite. Ditintills hade de flesta kon-

Icrcnscr i princip uteslutande behandlat tilliimpad

acridologi. dvs griishoppsbekiimpning med be-

slaktad prohlematik. Att det sedan skulle ta mer

lin 25 ir innan m<itet blev av hade nog inte initia-

tivtagaren Baccio Baccetti tdnkt sig.