COMMENTARY PAPER

Insect-truffle interactions – potential threats to emerging industries?

Aleksandra Rosa-Gruszecka1, Alan C. Gange2, Deborah J. Harvey2, Tomasz Jaworski1 , Jacek

Hilszczański1, Radosław Plewa1, Szymon Konwerski3, Dorota Hilszczańska4

1 Department of Forest Protection, Forest Research Institute, Sękocin Stary, Braci Leśnej 3,

05-090 Raszyn, Poland

2 School of Biological Sciences, Royal Holloway, University of London, Egham, Surrey

TW20 0EX UK

3 Natural History Collections, Faculty of Biology, Adam Mickiewicz University, Umultowska

89, 61-614 Poznań, Poland

4 Department of Forest Ecology, Forest Research Institute, Sękocin Stary, Braci Leśnej 3, 05-

090 Raszyn, Poland

Corresponding author: A.C. Gange, address above

E: a.gange@rhul.ac.uk

Tel. +44(0)1784 443188

Fax +44(0)1784 414224

Word count: 4,401

Running title: Insects and truffles

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ABSTRACT

Truffle harvests are declining in Europe, due to droughts, and this offers an opportunity for

production to be developed in countries such as the UK and Poland, where rainfall tends to be

higher. Drier Mediterranean summers seem to be associated with a decrease in the harvest of

the Périgord truffle (Tuber melanosporum) in Spain, France and Italy. However, other

species, for example the Burgundy truffle (T. aestivum) offer opportunities for production in

the more temperate environments north of the Alps. Truffles across Europe can be infested by

insect larvae, seriously reducing their economic and culinary quality. Here, using a

combination of literature sources and a field survey, we present a commentary on insects

attacking truffles, aiming to highlight those species that could be potential pests in the British

and Polish emergent industries. There is a remarkable disparity in coincidence of records of

insects and truffles in these countries, yet a survey in Poland confirms that insects can be

abundant. We discuss reasons for this disparity and suggest that biochemical methods could

easily be developed for detection of the truffles and their attackers.

Introduction

Truffles are hypogeous fungi belonging to the Pezizales, mostly in the genus Tuber, and

comprise a large group of ectomycorrhizal fungi growing in symbiosis with the roots of

several vascular plant species (angiosperms and gymnosperms). The fruit body of these fungi

is a subterranean complex apothecium, commonly known as the truffle. The geographic

distribution of truffles mainly covers the temperate zones of the northern hemisphere, with at

least three areas of genetic differentiation in Europe, South East Asia and North America

(Pomerico et al., 2006). So far, seven species of Tuber have been reported from Poland,

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namely T. mesentericum (Ławrynowicz, 1999), T. aestivum, T. excavatum, T. rufum

(Hilszczańska et al., 2008), T. maculatum (Ławrynowicz, 2009), T. macrosporum

(Hilszczańska et al., 2013) and T. brumale (Merényi, et al., 2014). According to the Fungal

Records Database of the British Isles (http://www.fieldmycology.net/FRDBI/FRDBI.asp) all

7 species have been recorded in the UK, though none have many attributed records. That with

the most is T. aestivum with 110 records, but to put this into perspective, the fungal species

with the most records (Hypholoma fasciculare) has 16,259 (as of 21 September 2016).

Economically, truffles are the most valuable non-timber products of forest ecosystems,

and are highly prized for their culinary qualities in countries such as France, Italy and Spain.

Highly desirable truffles (i.e. T. magnatum (white) or T. melanosporum (black)) may attract

remarkable prices, of around €2,000 - €3,000 kg-1, with the industry in Italy worth around

€400 million per annum (Büntgen et al. 2012; Pieroni, 2016). This may be the primary reason

why a truffle industry is emerging in countries such as the UK and Poland. However, it may

also be due to the decline of harvests of the highly-prized black truffle (T. melanosporum) in

its main habitats due to increased frequency of droughts (Büntgen et al., 2011; 2012; 2015).

Although neither T. magnatum nor T. melanosporum have been found in the UK or Poland,

two of their species (T. aestivum and T. brumale) are commercially traded in countries such as

Spain and Hungary (Martin-Santafe et al., 2014; Stobbe et al 2013). Indeed, recent evidence

suggests that T. aestivum in particular may be found in suitable areas north of the Alps, such

as Germany, and even as far north as southern Sweden and Finland (Stobbe et al., 2012;

2013). The first cultivated specimen of T. aestivum was found in England in March 2015

(http://www.bbc.co.uk/news/science-environment-31826764 ) and in Wales in July 2016

(http://www.itv.com/news/wales/2016-07-25/first-ever-cultivated-truffle-harvested-in-wales/).

In Poland, three truffle orchards (with T. aestivum) have been established and maintained by

the Forest Research Institute, one of which is productive (Hilszczańska et al., 2008;

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Hilszczańska and Sierota, 2010, Hilszczańska, 2016). Therefore, there is great economic

potential for the emergent industries in more northerly countries to fill gaps in the European

market, and it is timely to identify any problems that might reduce their potential in future.

Ecologically, these fungi are of considerable importance because of the benefits of the

mutualistic association they provide to their host plants (Pacioni and Comandini, 1999). In

addition, the relatively long-lived fruit body provides a food source for invertebrates and

vertebrates (Johnson, 1996; Blackwell, 2005). Some species of truffles, e.g. T. magnatum, T.

melanosporum and T. aestivum, have the high culinary value because of their aroma (Mello et

al., 2006) and in natural habitats, the volatiles produced are essential for attracting animals

that spread the spores (Fogel and Trappe, 1978). However, some animals have evolved a

capacity for feeding on truffle sporophores. These belong to various taxonomic groups and

are termed ‘hydnophagous’ (Pacioni, 1989), from Greek ‘hydnon’, truffle, and ‘phagous’,

eating. For certain animals, such as mammals (rodents, deer, boars), birds and slugs, truffles

are a valuable part of the diet (Johnson, 1996; Vernes et al. 2015), yet for several species or

genera of Arthropods, mainly in the Coleoptera and Diptera, the fungi may represent their

complete diet (Pacioni et al., 1995; DiSanto, 2013). Indeed, there is much anecdotal evidence

that truffles may be discovered by searching for the flies that oviposit within the fruit body,

though this is a laborious and unpredictable procedure (Blackwell, 2005). Scattered through

the literature are occasional reports of truffle sporocarps being infested by insect larvae, thus

reducing their marketable value considerably (Ciampolini and Suss, 1982; Martin-Santafe et

al., 2014). Our aim here is to provide a survey of the insects that may be associated with

truffles in two countries, which as well as being ecologically interesting, highlights potential

problems for the emerging truffle industry.

Survey methods

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We present a survey of known host associations in each country, using databases and field

sampling. The UK insect fauna is relatively well recorded and we used national distribution

data, available through the National Biodiversity Network Gateway (https://data.nbn.org.uk/).

The UK National Grid divides the country into 2,500 10 km x 10 km squares and records are

provided at this scale. We extracted a list of the 10 km x 10 km squares in which T. aestivum

has been recorded and compared these with 10 km x 10 km records of the principal insect

species associated with truffles (described below).

National record data for Poland are far less developed, but we extracted data for fungi and

insects from the Universal Transverse Mercator (UTM) 10 km x 10 km grid

(http://baza.biomap.pl/pl/db). The Polish UTM divides the country into 3,384 10 km x 10 km

squares (Iwan et al., 2012). These were supplemented by surveys in four geographical regions

in Poland: Nida Basin, Przedbórz Upland, Miechów Upland and Chełm Hills (Table 1).

Records were obtained using two methods of collecting insects associated with truffles. In

2012-2014 Tuber spp. fruit bodies inhabited by adults and larvae were collected and larvae

reared to adult. Information on sampling effort is given in Table 2.

In addition, insects were also collected in two adjacent regions of Nida Basin and

Miechów Upland with traps installed in natural habitats of T. aestivum. A total of 24 modified

funnel traps were used in both localities. To minimize collection of non-target invertebrates

(e.g. Carabidae, Silphidae) or small vertebrates (lizards, mice), traps were buried in soil with

the upper edge of the funnel left a few centimeters above the ground, and were covered with a

plastic roof placed 2 cm above the funnel. To preserve collected insects, a container filled

with 200 ml of ethylene glycol was put underneath the funnel. Each trap was baited using a

small piece of T. aestivum mature fruit body, placed inside a 2 ml perforated container that

was installed under the trap cover. Traps were emptied every third week from mid-July to

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early October 2012. Beetles were identified by S. Konwerski and flies (Diptera) were

identified by A. Woźnica.

Truffle insects and their distributions

The coleopteran fauna associated with truffles is mainly represented by the beetle Leiodes

cinnamomea (Coleoptera: Staphylinoidea) (Arzone, 1970; 1971). Adult females of the species

are attracted by truffle volatiles in its early stage of growth, but not when the fruit body is

mature (Hochberg et al., 2003). The beetle appears to be specific to the genus Tuber,

particularly T. melanosporum, with some records from T. aestivum and T. excavatum (Fogel

and Peck, 1975; Pacioni et al., 1991; Bratek et al., 1993) and completes its life cycle in or

adjacent to the fruit body (Arzone, 1970; Newton, 1984). Nevertheless it can cause extensive

damage to the truffle (Fig. S1). In the UK, L. cinnamomea is considered hard to find

(Blackwell, 2005) and is designated as Nationally Notable, having been recorded in only 25

of the 2,500 10 km x 10 km squares of the National Grid (https://data.nbn.org.uk/ (accessed

21/9/2016)). Its most likely host in the UK, T. aestivum, has been recorded from 40 of the 10

km x 10 km squares, yet in only 3 squares are there coincidental records of beetle and truffle.

This represents just 12% of the recorded beetle distribution and 7.5% of squares with truffle

records. Given that the insect is host specific, one would expect that all beetle records would

coincide with those of the truffle, but instead there is a great discrepancy in records. This is

likely due to the well-known bias that exists within such data bases (e.g. Ward, 2014), but

with truffles in a country like the UK it is likely to be particularly acute. As the fungus is

highly prized, locality records are very unlikely to be advertised by those seeking the fruit

bodies for commercial purposes, creating a highly biased distribution. Entomologists are more

likely to present their records, as they are far less likely to have a vested interest in truffle

collection. Thus, the available data suggest that the insect may have little impact on the

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industry, but this is subject to the serious bias aforementioned. It is likely that L. cinnamomea

is considerably more common than thought and should not be dismissed as a potential pest

species.

In Poland L. cinnamomea is known from 15 squares of the UTM 10 km x 10 km grid

(http://baza.biomap.pl/pl/db (accessed 21/9/2016)). However, none of these localities are

coincident with known Tuber spp. sites. The field survey (Table 1) showed that larvae of this

beetle can infest ascocarps of T. aestivum and T. exacavatum. However, considerably fewer

larvae were found in T. excavatum. Along with L. cinnamomea another leiodid beetle, L.

oblonga, was found, both as adults and larvae, in T. aestivum fruit bodies. Other hosts of this

species’ larvae were T. excavatum and T. rufum (Table 1). Koch (1991) reported L. oblonga

from truffles, but without distinguishing the fungal species. L. oblonga has been recorded

from 16 10 km x 10 km squares in the UK, yet only one of these coincides with T. aestivum.

The results here seem to be the first report on L. oblonga associations with truffles. L. oblonga

is very similar to L. cinnamomea but correct identification of both species is possible based on

the analysis of the male genitalia (Nunberg 1987). Therefore, further studies on Leiodidae

associated with truffles should take into consideration the problem of similarity of both

species and molecular methods may be best employed to separate them.

Other species of beetle in the same family as L. cinnamomea in the UK include

Agaricophagus cephalotes and Colenis immunda (Horsfield, 2002). While these species are

known to attack T. aestivum elsewhere (Bratek et al., 1993; 2010), there is no information

available on their biology in the UK. A. cephalotes is very rare (just seven 10 km x 10 km

records), while C. immunda is more common (57 10 km x 10 km records, of which three

coincide with T. aestivum). C. immunda was also reared from fruit bodies in the Polish field

survery (Table 1). However, this beetle appears to have many above-ground fungal hosts

(Schigel, 2011), and so is likely to pose little threat to truffle cultivation.

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The fruit bodies of truffles are also commonly inhabited by various species of Diptera,

mainly of the genus Suillia (Ciampolini and Suss, 1982; Krivosheina, 2008). The adult

females fly close to the soil surface and lay their eggs on the ground, above the truffle fruit

bodies, so that the larvae can easily reach them on hatching (Talou et al., 1990). Larval

feeding can cause extensive damage (Fig. S2). Martin-Santafe et al. (2014) and Duaso (2012)

report infestation of fruit bodies of T. aestivum and T. melanosporum by another fly,

Helomyza tuberivora (= Suillia gigantea) but this species does not seem to occur in the UK.

Instead, Suillia affinis (which appears to be a senior synonym of Helomyza affinis) and S.

pallida do occur, S. affinis having been recorded from 99 of the 10 km x 10 km squares and S.

pallida from 58. Of the S. affinis squares, just four are coincidental with T. aestivum,

suggesting that either the same biased recording problem exists or that the fly is not host

specific (Baehrmann and Adaschkiewitz, 2003). Notwithstanding the recording problem, the

four squares represent 5% of fly squares and 12.5% of truffle squares. A similar situation

exists with S. pallida where the figures are 3.4% and 5%, respectively. Thus neither species

may pose much of a threat to UK truffle species such as T. aestivum, even though they infest

these elsewhere (Papp, 1994; Baehrmann and Adaschkiewitz, 2003).

As in UK, the truffle fly S. gigantea has not been recorded in Poland, in contrast to S.

affinis which was reported from the southern part of the country (Ojców National Park)

(Woźnica and Klasa, 2009). Soils in this part are calcareous and conducive to truffle

development, so T. aestivum could be a potential host species. The fly was the most numerous

of all trapped insects (Table 1). Based on research of Lo Giudice and Woźnica (2013) we can

also speculate that since localities of S. gigantea and S. affinis overlap in such regions as

Tuscany, Lombardia and Umbria, S. affinis may be an indicator of truffle presence and a

potential pest in Poland.

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Chandler (2010) reported the association with Tuber for Diptera of the genus Cheilosia

(Syrphidae) and one species (C. soror) has been bred from truffles (Falk, 1991). This fly has

been recorded from 173 of the 10 km x 10 km squares in the UK. Perhaps of most interest is

that this species shows a very different coincidence with T. aestivum compared with the

previous insect species: 35% of the truffle squares also possess a record of this fly. While

there is virtually no data available on the biology of this species, it would certainly merit

investigation as a species of potential pest concern.

Truffle biochemistry

The chemicals given off by truffles contribute to their characteristic aroma and gives them

their high monetary value (Costa et al., 2015). This suite of volatiles gives each a species-

specific profile, which changes over time. This has been linked to maturation of the fruit

body, as well as environmental factors and more recently to genetic variability (Splivallo et

al., 2012). A single fruit body produces 20-50 volatile organic compounds (Culleré et al.,

2010; Splivallo et al., 2011), but those considered to be biologically important include

dimethyl sulphide, which attracts dogs and pigs, and eight carbon-containing volatiles, which

attract the two main insect species associated with truffles, L. cinnamomea and S. pallida

(Talou et al., 1990; Splivallo et al., 2012).

Demand for such a rare and expensive crop in the UK and Poland can only be realised if

the truffles are both of good quality and pest-free. Recent chemoecological research has,

therefore, centred around identification of the exact chemical profiles emitted by the fruit

bodies and how they change over time. This information can then be used in the development

of devices to monitor both the quality of the truffles i.e. electronic noses (Costa et al., 2015;

Pennazza et al., 2013) and pest control in truffle harvests (Hochberg et al., 2003).

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The use of volatiles given off by the fruit bodies of truffles to reduce infestation by insect

species has engendered remarkably little interest. To date, this work has focused on L.

cinnamomea, and its attraction to both the volatiles produced by truffles and those produced

by the beetle itself (Hochberg et al., 2003). These authors showed that neither sex is attracted

by ripe truffle odours, but that females are attracted to immature truffles and males to

pheromones produced by females. To date, these observations have never been examined

experimentally. It could be critical in aiding the emergent industries to protect their harvests

against pest insects through the development of attractant traps that would divert the beetles

from the fruit bodies. However, this presents an intriguing philosophical dilemma; if the

beetles are as rare as national records suggest, then one could question the ethics of

developing traps that kill such rare, pest insects. There is a clear need to develop traps that

catch live insects, in order to accurately determine population sizes and distributions and

thereby address this dilemma.

Conclusions and future perspectives

Truffles are highly prized and their economic value is dependent not only on the aromas they

emit, but also on the fruit bodies being free of insect larvae. Truffle harvests have shown

notable declines in parts of Europe, and this offers important economic opportunities in

countries such as the UK and Poland to fill market gaps. It is important that these emergent

industries do not fail due to poor material that is infested with insects. Here, we have tried to

highlight those species of insect most likely to become pests in this industry. Understanding

their ecology will enable us to determine whether the disparity in national records of insects

and their hosts is real or due to recorder bias. Prevention of attack by insects is important for a

truffle producer, but presents an ethical dilemma of whether such rare insects should be

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trapped and killed. The development of electronic noses could aid truffle harvesters, since

these will be consistent and immortal, unlike a pig or a dog.

Acknowledgments

We are deeply grateful to Dr Domizia Donnini (University of Perugia, Italy) for help with

specimens of Leiodes cinnamomea, Dr Andrzej Woźnica (Wrocław University of

Environmental and Life Sciences, Poland), Dr Cezary Bystrowski (Forest Research Institute,

Poland) for identification of Suilla affinis and Karol Komosiński (University of Warmia and

Mazury, Poland) for identification of Atheta dilaticornis. We also thank prof. Jerzy Borowski

(Warsaw University of Life Sciences, Poland) for providing literature.

This work was supported by State Forest Holding, project No. OR-2717/19/11, Forest

Research Grant No. 260102 and by the Polish Ministry of Science and Higher Education,

project No. 240309.

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Table 1. Insects associated with Tuber spp. in Poland (asterisks indicate number of specimens

reared from fruit bodies of Tuber, data without asterisks show number of specimens caught in

traps).

Insect species Tuber species Nida Basin

Miechów Upland

Przedbórz Upland

Chełm Hills

DipteraHeliomyzidaeSuillia affinis (Meigen, 1830) T. aestivum 2635 1875 3*

ColeopteraBolboceratidaeOdonteus armiger (Scopoli, 1772) T. aestivum 1LeiodidaeAnisotoma orbicularis (Herbst, 1792) T. aestivum 1Apocatops nigrita (Erichson, 1837) T. aestivum 1Colenis immunda (Sturm, 1807) T. aestivum 4* 1Fissocatops westi (Krogerus, 1931) T. aestivum 6 5Leiodes cinnamomea (Panzer, 1793) T. aestivum 33* 25*

T. excavatum 5*Leiodes oblonga (Erichson, 1845) T. aestivum 14+1* 10 22*

T. excavatum 1*T. rufum 7*

Leiodes polita (Marsham, 1802) T. aestivum 2Nargus velox (Spence, 1815) T. aestivum 1Ptomaphagus sericatus (Chaudoir, 1845) T. aestivum 141 3Ptomaphagus varicornis (Rosenhauer, 1847) T. aestivum 1Sciodrepoides fumatus (Spence, 1815) T. aestivum 2 1Sciodrepoides watsoni (Spence, 1815) T. aestivum 5 4PhalacridaeStilbus testaceus (Panzer, 1797) T. aestivum 7*StaphylinidaeAtheta dilaticornis (Kraatz, 1856) T. aestivum 1*

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Table 2. Number of Tuber spp. fruit bodies collected in Poland in 2012-2014.

Tuber species YearRegion

Nida Basin Miechów Upland

Przedbórz Upland Chełm Hills

T. aestivum2012 35 32013 125 1662014 484 368

T. excavatum2012 2362013 4272014 291 17

T. rufum2012 120132014 5

Total 1599 537 22

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Supplementary on-line material

Insect-truffle interactions – potential threats to emerging industries?

Aleksandra Rosa-Gruszecka, Alan C. Gange, Deborah J. Harvey, Tomasz Jaworski , Jacek

Hilszczański, Radosław Plewa, Szymon Konwerski, Dorota Hilszczańska

Fig S1 Damage to T. aestivum fruit bodies by larvae and adults of L. cinnamomea

Fig S2 Extensive damage to T. aestivum caused by Suillia larvae.