ISSN 0945-3954 Studia dipterologica 23 (1) 2016: 139–145

Diptera attracted by the truffle Tuber aestivum Vittadini and by dimethyl sulphide in Sweden

[Von der Trüffel Tuber aestivum Vittadini und von Dimethylsulphid angelockte Zweiflügler in Schweden]

by Ingemar STRUWE and Christina WEDÉN

Uppsala (Sweden) Uppsala (Sweden)

Abstract The ability of the truffle Tuber aestivum Vittadini and of dimethyl sulphide, its volatile organic compound, to attract Diptera has been studied in the field in Sweden. Both the truffle and the compound were found to attract identically and exclusively, especially of the Robineau-Desvoidy. of that genus indicated by their behaviour the locations of hypogeous truffles in nature. We conclude that dimethyl sulphide is the active compound attracting Diptera to T. aestivum in the field and that heleomyzid flies are specifically attracted. The main “truffle ” in SwedenSuillia is pallida (Fallén, 1820), although other Suillia as well as Tephrochlamys Loew and Tephrochlaena Czerny are involved. The mycetophilid Stigmatomeria crassicornis (Stannius, 1831) as well as the trichocerid saltator (Harris, 1776) are also attracted by dimethyl sulphide. Key words Heleomyzidae, Suillia, , Stigmatomeria, , Trichocera, Europe, Sweden, Truffle,Tuber aestivum, semiochemical, dimethyl sulphide

Zusammenfassung Die Trüffel Tuber aestivum Vittadini und Dimethylsulfid, einer ihrer flüchtigen organischen Inhaltsstoffe, wurden in Feld- versuchen in Schweden auf die Fähigkeit hin untersucht, Zweiflügler anzulocken. Sowohl die Trüffel als auch die chemische Verbindung erwiesen sich gleichermaßen und ausschließlich attraktiv für Heleomyzidae, insbesondere aus der Gattung Suillia Robineau-Desvoidy. Die Fliegen dieser Gattung zeigten durch ihr Verhalten natürliche Standorte hypogäischer Trüffel an. Daraus wird geschlussfolgert, dass Dimethylsulfid der aktive Inhaltsstoff ist, mit dem T. aestivum in der Natur Zweiflügler anlockt und dass die Substanz besonders attraktiv auf Heleomyzidae wirkt. Die bedeutendste „Trüffelfliege“ in Schweden ist Suillia pallida (Fallén, 1820), obwohl auch andere Arten von Suillia als auch von Tephrochlamys Loew und Tephrochlaena Czerny beteiligt sind. Die Pilzmücke Stigmatomeria crassicornis (Stannius, 1831) sowie die Wintermücke Trichocera saltator (Harris, 1776) werden auch von Dimethylsulfid angezogen. Stichwörter Heleomyzidae, Suillia, Mycetophilidae, Stigmatomeria, Trichoceridae, Trichocera, Europa, Schweden, Trüffel, Tuber aestivum, Botenstoff, Dimethylsulfid

Introduction

The truffle Tuber aestivum Vittadini is widely distributed in Europe. Its most northern known locality is the Baltic island Gotland, Sweden (Wedén 2004). Since the late 1990s, scientific knowledge and public aware- ness of the natural distribution of truffles in Sweden has developed and also caught increasing interest from those involved in Scandinavian gastronomy. A market for truffles has been established in Sweden since then, involving truffle hunting with trained dogs in the wild and truffle cultivation (Wedén et al. 2009). A Swedish truffle hunter noticed the presence of hovering flies in spots where she would also find truffles (Eva Pettersson, pers. comm.). After learning about the French method of finding mature specimens of truffles by observing the

© Senckenberg Gesellschaft für Naturforschung, 2017 139 Struwe & Wedén: Diptera attracted by the truffle Tuber aestivum and by dimethyl sulphide hovering behaviour of certain flies on the ground, she started to use this method successfully to find mature truffle specimens in her local truffle grounds. A truffle is a fungal life-form defined by its hypogeous fruiting bodies. The ability of active spore dispersal has been lost so the fungus is instead dependent on spore vectors, perhaps including . At full spore maturity, truffle fruiting bodies emit an array of volatile organic compounds (VOCs) that attract mammals such as mice, squirrels and boars to unearth and eat them. The spores are then efficiently spread in faecal pellets (Trappe & Castellano 1991). The truffle life-form has developed multiple times in fungi (Trappe et al. 2001), and volatile and non-volatile components are being studied to understand the nature of the fungus-animal interactions. Truffle VOCs have shown potential for interaction with , plants, other fungi and bacteria (Splivallo et al. 2011). Due to the gastronomic, and hence economic, interest in certain Tuber spp., most studies on truffle VOCs have been done on species belonging to this genus in the Ascomycota (Splivallo et al. 2011). Although common in many species, the relative quantities of VOCs give each truffle species a unique, species-specific scent at full spore maturity. The characteristic aroma of mature Tuber fruiting bodies is to a large extent due to small molecules, often sulphur-containing hydrocarbons, often comprising alcohol, aldehyde and/or ketone functional groups (Splivallo et al. 2011). In T. aestivum, the most prominent compounds producing its scent, as identified by gas chromatography – mass spectrometry (GC-MS) of headspace solid-phase microextraction, are dimethyl sulphide, 2-methyl propanal, 2-methyl butanal, 3-methyl butanal, 2-methyl-1-propanol, 2- methyl-1-butanol and 3-methyl-1-butanol (Dìaz et al. 2009). Culleré et al. (2010) on the other hand, used GC-olfactometry to identify the VOCs most important to the human perception of the aroma of T. aestivum, and these were dimethyl sulphide, dimethyl disulphide, methional, 3-methyl-1-butanol, 1-hexen-3-one and 3-ethylphenol. Although similarities exist between the two studies, the differences in identified compounds are evident and may be accounted for by the specific sensitivity of the human nose to sulphur-containing VOCs. In the middle of the 19th century Goureau (1852) and Laboulbène (1864) respectively reared insects from truffles and identified a list of the Diptera in truffles which was little changed for the next hundred years. They verified the fly family Heleomyzidae and its genusSuillia as important members of the truffle-associated fauna. Hackman & Meinander (1979) in Finland widened the perspective by rearing Diptera from non-hypogeous macrofungi and found that the genus Suillia was richly represented even there. Bratek et al. (2001) reared insects from truffle sporocarps in Hungary and identified beetles and flies mainly to family level. Again the heleomyzid genus Suillia was strongly numerically dominant, especially Suillia pallida (Fallén, 1820). The genus Suillia seemed to have a specificity for Tuber species, as it was not reared from any other truffle genus. Rearing records in Britain of (Meigen, 1830), S. pallida, S. affinis (Meigen, 1830) and (Loew, 1862) from truffles were reported inC handler (2010). Suillia species also became commonly known among Central European truffle hunters as “the truffle fly”, but scientific confirmation of this is still lacking (Chandler 2010). Field studies in Sweden (Tjernby 2003) designed for spore dispersal research revealed Suillia species in some traps baited with the truffle Tuber aestivum, but no further identification was made. García-Montero et al. (2004) in Spain sampled flies by netting in habitats with known high abundance of the truffle Tuber mesentericum Vittadini and identified them all as (Meigen, 1830), a species also frequent in truffle regions in France C( outin 1989). Talou et al. (1990) studied the ability of a number of VOCs from truffles Tuber( melanosporum Vittadini) to attract mammals such as dogs and pigs; the only buried sample reliably localized contained dimethyl sulphide (DMS). Pacioni et al. (1991) tested the attracting ability of different truffle VOCs and found that DMS was superior in attracting insects. Identification was however only made to level in Diptera and family level in Coleoptera. In an excellent research review Splivallo et al. (2011) stated that in order to confirm that DMS is the actual attractant in nature, behavioural assays with real truffles should be conducted.

Aim of study We designed a study in order to identify the fly species attracted by T. aestivum in nature in Sweden, and to evaluate the role of DMS in attracting flies under field conditions. Comparing the fly fauna attracted by DMS

140 Studia dipterologica 23 (1) 2016: 139–145 traps and truffle traps, we would try to find further evidence that DMS is the active semiochemical of truffles. Through field observations, we wanted to identify fly species with verified truffle-indicating behaviour.

Materials and methods The study was conducted on the Baltic island Gotland in areas with well-known high abundance of T. aestivum during two separate weeks in autumn 2014: late September and early November. In addition the third activity below was performed during October. These calendar periods were chosen to reflect expected higher fly activity and truffle maturation, respectively. Four different activities were performed. 1. Sealed 10 litre plastic boxes with a narrow funnel entrance (Fig. 1) were placed in the terrain in two separate lines 200 metres apart, 10 boxes in each line and 50 metres between each box. In one line the boxes were baited with fresh mature truffle, and in the other line with partly sealed tubes containing DMS. The boxes were emptied daily and the entire yield preserved and morphologi- cally identified using the keys in Beuk (2012), Dahl (1966), Gorodkov (1989), Hedström (1995), Krzeminska (1999), Peeters & Oosterbroek (2014), and Withers (1987). 2. Field observations were carried out in other truffle areas. The ground was thoroughly scruti- Fig. 1: Trap box with truffle or dimethyl sulphide. Photo: I. Struwe nized for typical fly behaviour such as hovering close to the ground or landing for oviposition. Any such flies observed were to be collected, preserved and identified. Indicated spots were to be marked with flags for later determination of truffle presence or absence by the use of a specially trained truffle dog. 3. In a parallel activity in another area, an experienced truffle hunter sampled flies with typical behaviour and verified truffle occurrence or absence by immediate digging. These samples were also preserved and identified. 4. As a blind control, a Malaise trap was established in a representative area adjacent to the trap lines during both study weeks. The material obtained was identified to family (Diptera) or order (other insects).

Results The weather conditions turned out to be very harsh during the study periods in September and November: rainy, windy and chilly. Nevertheless, a good catch of flies was obtained in the baited traps especially in November (Tab. 1) and in the Malaise trap especially in September (Tab. 3). Field watching for truffle-indicating flies was unsuccessful in September but successful in October and November (Tab. 2). Exclusively Heleomyzidae flies were caught in the truffle and DMS traps as well as in the field netting activity on verified truffle growing spots (Tab. 1). Nearly all belonged to the genusSuillia with S. pallida (Fallén, 1820) numerically dominant in both the traps and the field nettings. Other recorded Suillia species were S. humilis (Meigen, 1830), S. affinis( Meigen, 1830), S. vaginata (Loew, 1862), S. fuscicornis (Zetterstedt, 1847), S. parva (Loew, 1862) and S. laevifrons (Loew, 1862). Tephrochlamys flavipes (Zetterstedt, 1838) and Tephrochlaena halterata (Meigen, 1830) were also represented by a few specimens. Two specimens of bifurcata (Fallén, 1810) were found in the DMS traps as well as some fungus gnats (Mycetophilidae) and winter gnats (Trichoceridae) and some parasitic wasps (Ichneumonidae). The fungus gnats were all Stigmatomeria crassicornis (Stannius, 1831) and the winter gnats Trichocera saltator (Harris, 1776), both species equally abundant in the traps.

141 Struwe & Wedén: Diptera attracted by the truffle Tuber aestivum and by dimethyl sulphide

Tab. 1: Species and numbers of insects trapped and observed.

Insect taxon Specimens Specimens Specimens with in in truffle-indicating truffle trap DMS trap field behaviour DIPTERA Heleomyzidae (Meigen, 1830) 3 2 (Zetterstedt, 1847) 1 Suillia humilis (Meigen, 1830) 5 8 (Loew, 1862) 1 Suillia pallida (Fallén, 1820) 7 129 47 Suillia parva (Loew, 1862) 1 Suillia vaginata (Loew, 1862) 2 1 Tephrochlaena halterata (Meigen, 1830) 1 Tephrochlamys flavipes( Zetterstedt, 1838) 1 1 (Fallén, 1810) 2 Mycetophilidae Stigmatomeria crassicornis (Stannius, 1831) 5 Trichoceridae Trichocera saltator (Harris, 1776) 4 HYMENOPTERA Ichneumonidae 4

Tab. 2: Observation spots with suspicious fly behaviour (confirmation rates).

Observation Number of Spots with truffle Spots with Percent month observation spots confirmed truffle not confirmation confirmed September 1 – 1 (by man) – October 8 8 (by man) – 100 November 26 18 (by dog) 8 69

Truffle presence at locations with suspicious fly behaviour was confirmed in every case of immediate human digging and in 69 % of cases involving a search with a dog the following day (Tab. 2). Trap results and field netting results agreed well with respect to fly species recovered. Notably few Heleomyzidae were caught in the Malaise trap during both periods (Tab. 3), the species represented being Suillia laevifrons, Suillia vaginata, Tephrochlaena halterata, Tephrochlamys flavipes and Eccoptomera filata Loew, 1862. In the Malaise trap material Heleomyzidae constituted 0 % of the September and 7.5 % of the November total specimen number in sharp contrast to the baited box traps. The wide variety of other fly families and orders in the Malaise trap was not reflected in the truffle or DMS traps except for Mycetophilidae, Trichoceridae and Ichneumonidae.

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Tab. 3: Malaise trap results, specimens trapped.

Insect taxon September November COLLEMBOLA 20 0 DIPTERA 224 62 0 3 1 0 1 0 10 0 Heleomyzidae 0 6 10 0 50 6 Sarcophagidae 0 2 50 4 Syrphidae 2 0 Lower Diptera [‘’] 100 41 HEMIPTERA: Aphidoidea 20 0 HYMENOPTERA 100 18 THYSANOPTERA 10 0

Discussion The results clearly point out the heleomyzid genus Suillia as the outstanding truffle-attracted fly genus in Sweden, and dimethyl sulphide (DMS) is obviously the strong attracting semiochemical. The Suillia species were also responsible for nearly all truffle-indication behaviour. Among these, Suillia pallida was numeri- cally dominant. Suillia is represented in Sweden by 27 species, most of which are mainly mycetophagous. They are also notably cold-resistant and are often observed on snow in winter. Thus they are well suited for using the late-maturing truffle T. aestivum as a larval food source; competition is also low in November. The truffle fungus itself, requiring active vector mediated spore dispersal, may benefit from the fly larval develop- ment in its hypogeous sporocarp for local spore dispersal in the soil (Arzone 1970, Tjernby 2000). Whether hatched flies carry spores or not has not yet been investigated. The nearly exclusive Suillia dominance in our study agrees with field observations in Spain G( arcía-Montero et al. 2004) and France (Coutin 1989). Moreover, our study links the behaviour of the fly directly to the sub- terranean location of the truffle fruiting body as well as to one of its volatile organic compounds, DMS – and also identifies the spectrum of Suillia species simultaneously involved. Besides Suillia also Tephrochlamys flavipes is mentioned in earlier surveys (Jakovlev 1994, Chandler 2010), and this is confirmed in our study. The occurrence of Suillia laevifrons in DMS trap is notable since it has been reported to have phytophagous larvae boring shoots of Luzula (Bland & Rotheray, 1996). The families Mycetophilidae and Trichoceridae share the same properties of cold resistance and winter activ- ity, but although abundant in the truffle area as reflected by the Malaise traps, there were only few specimens found in DMS traps and none in the truffle traps.B ratek et al. (2001) could not detect any mycetophilids reared from truffles but there are other reports in the literature on the mycetophilidStigmatomeria crassicornis reared from truffles (Edwards 1925) and also damaging cultivated truffles in North America C( handler 2010). Our record of Stigmatomeria crassicornis in DMS traps adds support to its relation to truffle. Trichoceridae has not previously been reported from truffles although at least five species of this family are known to also develop in

143 Struwe & Wedén: Diptera attracted by the truffle Tuber aestivum and by dimethyl sulphide

fungi: Trichocera annulata Meigen, 1818, T. hiemalis (De Geer, 1776), T. regelationis (Linnaeus, 1758), T. rufescens (Edwards, 1921) and T. saltator (Harris, 1776) (Hackman & Meinander 1979; Chandler 2010). Our finding ofT. saltator attracted by DMS is interesting and may indicate that truffle is a possible substrate for its larval development. Parasitoid wasps (Ichneumonidae) trapped in the DMS boxes provide an interesting find. It may suggest that they use the same semiochemical for locating the host, a well-known phenomenon in for example forest ecology. Further studies are necessary in this matter.

Acknowledgements

We thank the landowners Ulf Gustafson and Christina Rydegran (Lärbro, Sweden), Ingvar Jakobsson and Ylva Rudin (Eskelhem, Sweden), Ulla and Claes Berglund (Rute, Sweden) for generously offering their land for our study; the truffle hunters EvaP ettersson (Etelhem, Sweden), Olof Thomson (Östergarn, Sweden) and Jane Ekström (Visby, Sweden) for skilful work in the field; ÅkeS truwe (Stockholm, Sweden) for invaluable field assistance; Maria Backlund (Uppsala, Sweden) for participation in the early planning of the project; Peter Chandler (Melksham, United Kingdom) for important remarks on the manuscript and confirmation of the identity of Stigmatomeria crassicornis; Yngve Brodin (Stockholm, Sweden) for the identification of Trichocera saltator; the Entomological Society of Stockholm for financial support and lastly the lagotto romagnolo dogs Pucko, Bruno and Darwin for impressive truffle detection ability.

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Authors’ addresses

Ingemar Struwe Stenbrohultsvägen 111 75758 Uppsala Sweden E-mail: [email protected]

Christina Wedén Division of Pharmacognosy Department of Medicinal Chemistry Uppsala University BMC Box 574 75123 Uppsala Sweden E-mail: [email protected]

The paper was accepted on 10 June 2017. Editum: 28 December 2017.

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