Attraction of Phyllopertha horticola (Coleoptera, ) 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 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 ' 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.) arc attracted/excited primarily by odour, or has repeatedly been observed to visit flowers of whethcr visual and tactile cues are also necessary Ophrys L., sectioll Fuciflorae Rchb.f. (Kullen- to elicit the complete sequenced behavioural re- berg 1973, Warncke & Kullenberg 1984. Borg- sponses leading to pollination by pseudocopula- Karlson 1987). The often white or cerise sepals of tion. Oplrrys flowers are similar in coloration to th€ The purpose of this study was to experimentally beetles' food plants Cisrr.r (Cistaceae) and Crata- examine, using behavioural tests, the role ofolfac- egus (Rosaceae). It has been suggested that these tory stimuli in the pollination relationship betwe- colours may act as visual cues encouraging both en the beetle Phyllopertha horticola and the spe- male and female beetles to visit and forage on cies Ophrys holocericea, O. scolopax Cav. and O. tN Ophr,r,s flowcrs and thcre bv incidentally pollinate t ent h re d i nif e r a illd. them (Ferlan in lit. to Kullenberg. Kullenbcrg 1961). However, recent observations (Forster, Materials and methods Paulus, in lit. to Kullenberg 7976,7982,1983) re- vealed that P. horticola malesbehave as ifsexually The study took place on Oland, one of the large is- stimulated when visiting O. holocericea (Btrm. lands in the Baltic Sea closc to the mainland of fil.) Greuter l: O. fuciflora (Cranz) Moenckl southcrn Sweden. Odour attraction tcsts were flowers. Other scarabaeid beetles, such as Tropi- performed from end May to mid Junc 1983-85 on nota squalida Scop. and Oxythyrea funesta Poda, a field population of beetles inhabiting the border also visit and pollinate various Opftrys species in of a grass-lined gravel road surrounded by hazel- the section Fuciflorae (Ktsllenberg 1961). bushes (Corylus avellana L.). It has not been established whether the beetles To test the beetles attraction to different 106 Anns- Karitt l)org-Korlson

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 h i.+o 66p. 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, Fig. l. The odour-guidcd rcsponses of Pryllopertha hor- were noted. Results were evaluated statistically ricola male beetles are divided into four behaviour tvpcs using the chi-square test for 2 x 2 contingency ta- based on increasing degrees of attraction/excitation: ( l) blcs. For each test sample, the sum of behaviour p : uninlluenced passage, (2) q.i. : quick inspection, types (h.i. (3) h. i. : hovering inspection and w.i. = walking inspec- 3 and 4 [hovering ), walking-inspection tion, (1) h.i.-a.cop. : hovering inspection followed by (w.i.), and alighting rcsponscs on the dummy "copulation attempt" or w,i. q,cop. : walking inspec- (h.i.-a.cop., w.i.-a.cop.)], i.c. thc strongly at- tion folkrwed bv "attempted copulation", The tracted beetles (Fig. l), was dividcd by the total are regar(lcd as strr',nglv attracted in the behaviour types 3 and 4. number of responses (N). Portions of thc be- havioural sequences were filmed using a Paillard Det dottinducerade beteendet som trddgirdsborrehanar 16 utfdr vid attrappen Ar uppdelat i fyra olika typer med sti- Bolex mm film camera. gandc attraktivitctsgrad: (l) 2 : passage, (2) S.i. = Voucher beetle specimens are deposited at the snabb inspektion, (.3) n.i. = sidsvingande eller r,r,.i. = Ecological Research Station of Uppsala Univer- krvpandc inspcktittn. (1) h.i.--a.cop. eller w.i.-a.cop. = sity on Oland. and nomenclature of sidsvlngandc cllcr krypande inspektion som slutar med fiirsitk till kopulation pe artrappen. Insekterna iir starkt Ophrys is based on Nelson (1962), sce also Borg- attraherade om de reagerar med beteendetyp 3 cllcr 4. Karlson ( 1987) and rcfcrcnces therein.

Results odours, eight different samples were presented to heetles within the confines of the swarm. Extracts In the mornings, male heetles started to walk were studied of Ophrys scolopax and O. lenth- whcn ambient temperaturc rcachcd 13"C (at 1.5 m rediniferu labella, and of female and inale /'ftyl- above ground) and to swarm at lzl-15"C. Swarm- lopertha horticola bodies. Fresh Oplrrys labella ing usually lasted for about three hours. Female were extracted in redistilled hexane (5 labclla/ml beetles were never seen swarming, bui were fre- hexane, Merck) for three days at 4'C and l-2 days quently observed feeding on leaves and grasses in old P. hortitolu bcctles were extracted in redistil- the vicinity. led pe ntanc (2 whole bodies/ml pentane, Merck) Only males were attracted to the dummies. The for 24 hours at 20'C. bchavioural rcsponses of malc bectles. both flying The following three authentic blends of com- and walking, to the dummies werc classified into 4 pounds identified in scents of Opftrys flowers were typcs as follows, listed in ordcr of increasing at- studied (volumes in pl; each blend diluted in 1.9 traction/excitation (Fig. 1). m.l hexane): Alcohol blend: l.hexanol 2, cis-3- 1. p passage: hexcn-l-ol 2. 1-octanol 10, l-hexadecanol l0; = uninfluenced by-passing without de- creasing flight speed or changing flight direction. Acid blend: pcntanoic acid 2, hexanoic acid 4, 2. q.i. = quick inspection: flight direction changed to- hcptanoic acid 10, octanoic acid 2, nonanoic acid wards dummy, sometimcs rcsulting in touch. 20; Ophrys tenthredinifera extruct blendi l-hexa- 3. r.i. : hovering inspection: mcandcring flight towards the odour source. hovering in front of it, sometimes fly- nol 2, ci.r-3-hexen- l -ol 2, 1-octanol 10, l-hexade- ing around it, always facing tho dummy. w.i. = walking canol 10, 6-methyl-5-hepten-2-one 6. pentanoic inspection: walking on ground (x in vcgetation towards acid 2, hexanoic acid 4. heptanoic acid l(). oc- clumml but without touch. Attractiort oJ Phyllopertha nales to Ophrys flowers 107

Tab. I . Respon sc s r 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 0.36* 0.19 64 10 O p hrys sc o lo pax labella extract 0.25- 0.10 40 8 Phyllopertha horticold nrale body extract 0.14 0.09 58 l0 P, horticolalemale body cxtract 0.13.* 0.06 70 8 O. tenthredinifera extract blcnd 0.12 0.10 50 l0 O. tenthre dinifero labclla extract 0.08 0.00 25 4 Acid blend 0.06 0.02 54 9 Hexane blank 0.05 0.02 56 10

' p

4. h .i.--a. cop. - hovering inspection followed by descent Discussion and walking on dummy.'fhe beetle performs move- ments that appear to represent a copulation attempt, Rcsults of the present odour-attraction Iests indi- tt.i.-a.cop. : walking inspection followed by contact cate that P. horticolq malcs are attracted to vol- with the durnmv and seeminglv attempted copulation. atile compounds prescnt in flower fragrances of Extracts of O. scolopax labella, which are domi the studied Op&rys species. White and cerise col- naled by the alcohols l-hexanol and 3-hexen-1-ol our cues were excluded in the experiments and the (Borg-Karlson 1987), proved to be highly attrac- beetles seemed to be only slightly, if at all, af- tive to P. horticola malcs (Tab. 1). The strongest fected by the visual stimulus of the dummy on attraction, howcvcr, was eliciled by the alcohol which odours were applicd. Only males were at- blend. This blend differed significantly in attrac- racted to the test-samplc dummies and, when tivity from the P. horticola female extract (Xr, highly excited, they performcd movements that p<0.05), the acid blend and the hexane blank (X'?, may be interpreted as "pseudocopulation". p<0.01), which released the two weakest re- Aliphatic alcohols seem to be of greatest impor- sponses. Attraction to the extract blend of O. tance in releasing this behaviour. tenthredinifera, which contained constituents of The body extract of female beetles was only both the alcohol and acid blends. was much lower weakly attractive to males which is in contrast to than that 1o O. sutktpux cxtracts, but similar to the results from a recent study of P- diversa those observed for hody cxtracts of male and of Watcrhousc where the female body extract was female P. horticola. When a highly excited male vcry attractive to conspecific malcs. This may, beetle responds to the alcohol blend the abdomen howcver, be influenced in part by the age of the is stretched out, the copulation apparatus fully fenrales. The dilution of the P. diversa female ex- clongated, and thc bcctle is eagerly grasping the tract was found to be of minor importance piece of velvct. This bchaviour must be inter- (Kawasaki & Tamaki 1985). preted as attempted copulation, and has not becn Thc aliphatic alcohols tcsted are not restricted observed when the beetle is feeding on its food in occurrence to the three Opftrys species exam- plants (Kullenberg pers. comm.). The very few ined here. but are also found in large amounts in approaches displayed by beetles towards the con- the fragrance emitted from O. lutea Cav. and O. trol hexane dummy suggest that the visual .ftrsca Link (Borg-Karlson et al. 1985, Borg- stimulus of the velvet might be negligible com- Karlson & Tengd 1986). However, to my knowl- pared to the olfactory stimuli of the tested odour cdge thcre are no observations of P. horticolavis- samples in eliciting copulation. iting these Oplzry.r specics, which may be lO8 Anna- Karin Borg- Karlson

explained by differences in the blooming times Ophrys sections Fuciflorae and Bombyliflorae as in- and habitats of diffcrent plant species and in sect mimetic altractants/ex.itanls. Scr. 27: 313-325. -Chem. P. swarming timcs ofdiffercnt beetle populations. Kawasaki, K. & Tamaki. Y. l9tl5. Evidence of the sex horticola belongs to a family that are known to re- attractant pheromone and activit! rhylhm in Phyl- spond to a variety of compounds (Nilsson A. pers. lopertha diversa Waterhouse (Coleoptera, (2): comm.). l-Hexanol and cis-3-hexen- 1-ot, both Scarabaeidae). Appl. Ent. Zool. 20 137 142. Kullcnberg. B. t961,- Studics in Ophrys pollination. present in the alcohol blend, are common in plants Zool. Bidr. Uppsala -j4: l--j.10. .51 plates. - and probably function as food attractants to many Kullenberg. B. 1973. Ncw ohscrvations on the pollina- insects. However. an odour induced sexual attrac- tion of Ophrys orchids [-. (Orchidaceae). - Zoon. tion to the aliphatic alcohol blend and to the Suppl. l:9-11. Kullenberg, B., Borg-Karlson. A.-K. & Kullenberg, Ophrys lab'ella extracts is strongly suggested by A.-L. 1984a. Field studies on the behaviour of the three observations: (l) beetles oriented towards Eucera nigrilabris male in the odour flow from flower the odourife rous dummy in the al\sence of white Iabellum extract of Ophrys tenthredinitera. - Nova or cerise colours (visual stimuli), (2) only males Acta Reg. Soc. Sci. Upsalicnsis. Ser. V:C,3r 79 l l0. Kullenberg. 8., Btiel. H., & Tkalci. B. 1984b. Uber- were attracted to thc dummies. (3) the behaviour sicht von Beobachtungcn iiber Besuche von Eucera of the descending nrales on the odouriferous und Tetralonia-Minnchen auf Ophrys-Bliiten (Or- dummv resembled the initiation of a copulation. chidaceae). - Nova Acta Reg. Soc. Sci. Upsaliensis. This behaviour was not initiated by the hexane Ser. V:C- 3: 27-.1U. Nelson, E. 1962. Gestaltwandel und Artbildung erdnen dummy (tactile stimuli not enough). am Beispiel der Orchidaceen Europas und der Mit- It is interesring to note that the pollination of telmeerlander insbesondere der Gattung Ophrys. Ophrys orchids in certain habitats are performcd 250 pp. 56 pl. 8 cartes. Chernex et Montreux. Warnckc. by insects other than bees. The importance of bee- K. & Kullenbcrg. B. 19x4. Uher.icht vt'n Beobachtungen Besuche von Andrena- und Colletes tles as pollen vectors and their flower preference cunicularius Mannchcn auf Ophrys BlLiten (Or- patterns in thc genus Ophrys, are not known. chidaceae). - -Nova Acta Reg. Soc. Sci. Upsaliensis, Their flower visits are most likely of low specific- Ser. V:C. 4l-55 ity. leading to interspecific pollen transfer and mav explain some of the hybrids found. The regu- Sammonfattning lar pollen vectors in the Opftrys section Fuciflorae are known to be males of the solitary bees Eucera Ophrys orkidd.er pollineras i huvudsak av solitdra and Terralonia (Kullenberg et al. 1984a,b). bi-, grdv- och dolkstckelhanar (Hymenoptera: Andrenidae, Anthophoridac, Megachilidae, Acknowledgements: I thank Prof. Bertil Kullenberg for Sphecidae och Scoliidae). Under senare ir har his enthusiastic support and valuable discussions con- dven minga observationer av besokande och pol- cerning this study. and tbr his excellent filmings of beetle behavioural sequences. I also want to thank Mrs Barbro linerande skalbaggar av familjen Scarabaeidae Holmlund for skilful technical assistance and Dr Anders rapporterats inom Ophrys sektionen Fuciflorae. Nilsson (Uppsala) and Dr Lcnnart Agren for valuable Dessa orkid6ers vita eller rddaktiga kalkblad har discussions and Dr Heidi Dobson for linguistic revision antagils ha stor betydelse insekternas of the manuscript. Financial support from the Swedish fdr oriente- Natural Science Research Council. the Bank of Swedcn ring till blommorna. Skalbaggens beteende pi den Tercentenarv Foundation. and the Axel and Margarct finludna blomliippen har heskrivits bide som Ax:son Johnson Fou ndation is gratefully acknowledged. ndringssrik och forsrik till kopulation. For att utvardera kemiska signalers betydelse References fdr skalbaggarnas orientering mot Oprrys-blom- morna, pa Borg-Karlson. A.-K., lJergstrdm, G., & Groth. L l98-5. -gjordes beteende-experiment i falt Chemical basis for the relationship between Ophrys Oland. Atta olika extrakt och substansblandning- orchids and their pollinators I: Volatile compounds ar testades pe triidgardsborrar (Phyllopertha hor- of O. lutea and O. fusca as insect mimetic aftractants/ llcola) vilka har observerats som pollinerare pi O. excitants. Chem. Scr. 25:283-294. Borg-Karlson,- A.-K. & Teng

Recensioner

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