Mites as matchmakers: Semiochemicals from host-associated mites attract both sexes of the parasitoid Lariophagus distinguendus

Article abstract of DOI:10.1023/A:1005484027559

The role of volatile chemicals used for mate finding was studied for males of Lariophagus distinguendus (Forst.), a parasitoid of the granary weevil Sitophilus granarius (L.). In bioassays that used a static four- chamber olfactometer, males were attracted by host feces, hexane extracts from host feces, and volatile extracts of the feces obtained by closed-loop stripping (CLS). On the other hand, volatiles emitted by unmated females did not elicit any preferences in males. Both sexes of L. distinguendus responded to a synthetic mixture of neral, geranial, neryl formate, and tridecane occurring in the investigated extracts. All compounds are common constituents of astigmatid mites that are often associated with possible hosts of L. distinguendus. In the system investigated, all main compounds found in CLS extracts from larval feces of S. granarius are due to the mold mite Tyrophagus putrescentiae (Schrank) that uses neral, geranial, and neryl formate as an alarm pheromone. The possible role of host-associated astigmatid mites in mate and host finding of L. distinguendus is discussed.

Full text of DOI:10.1023/A:1005484027559

Journal of Chemical Ecology, Vol. 26, No. 5, 2000
MITES AS MATCHMAKERS: SEMIOCHEMICALS FROM
HOST-ASSOCIATED MITES ATTRACT BOTH SEXES
OF THE PARASITOID Lariophagus distinguendus
JOACHIM RUTHER* and JOHANNES L. M. STEIDLE
Institut fu¨r Biologie, Freie Universita¨t Berlin
Haderslebener Str.9, D-12163 Berlin, Germany
(Received June 10, 1999; accepted January 1, 2000)
Abstract—The role of volatile chemicals used for mate finding was studied
¨
for males of Lariophagus distinguendus (Forst.), a parasitoid of the granary
weevil Sitophilus granarius (L.). In bioassays that used a static four-chamber
olfactometer, males were attracted by host feces, hexane extracts from host
feces, and volatile extracts of the feces obtained by closed-loop stripping
(CLS). On the other hand, volatiles emitted by unmated females did not
elicit any preferences in males. Both sexes of L. distinguendus responded
to a synthetic mixture of neral, geranial, neryl formate, and tridecane occur-
ring in the investigated extracts. All compounds are common constituents
of astigmatid mites that are often associated with possible hosts of L.
distinguendus. In the system investigated, all main compounds found in CLS
extracts from larval feces of S. granarius are due to the mold mite Tyrophagus
putrescentiae (Schrank) that uses neral, geranial, and neryl formate as an alarm
pheromone. The possible role of host-associated astigmatid mites in mate and
host finding of L. distinguendus is discussed.
Key Words—Granary weevil, Sitophilus granarius, parasitoid, Lariophagus
distinguendus, astigmatid mites, Tyrophagus putrescentiae, semiochemicals,
host finding, mate finding, neral, geranial, neryl formate, tridecane.
INTRODUCTION
For successful reproduction, parasitoids have to be able to locate their mates
and hosts. In most species, chemical cues are involved in mate- and host-finding
mechanisms.
In many parasitic Hymenoptera, mate finding is mediated by female sex
*To whom correspondence should be addressed.
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0098-0331 00 0500-1205$18.00 0  2000 Plenum Publishing Corporation
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RUTHER AND STEIDLE
pheromones. Often more than one component is supposed to be involved in
such pheromone systems, one serving for long-range attraction, others medi-
ating close-range courtship behavior (Quicke, 1997). However, if long-range
pheromones are missing, males have to rely on other chemical cues to locate
females. One possible strategy for males is to use the same chemical cues as
females use for host location because the probability of encounters with recep-
tive females increases at possible oviposition sites.
For host location, female parasitoids often use chemical cues from their
hosts, such as pheromones or feces (reviewed by Godfray, 1994; Quicke, 1997),
or indirect cues emitted by infested host plants after feeding (e.g., Dicke and
Vet, 1999; Steinberg et al., 1993; Turlings et al., 1991, 1995), or oviposition
of the herbivorous host (Meiners and Hilker, 2000). Volatiles emitted by host-
associated fungi also have been reported to attract female parasitoids (Greany et
al., 1977; Madden, 1968).
Females of the polyphagous parasitoid Lariophagus distinguendus use
volatiles emitted from larval feces of one of their hosts, the granary weevil,
Sitophilus granarius, for host location (Steidle and Scholler, 1997). However,
¨
the chemical structures of the compounds responsible for this phenomenon have
not been characterized.
In the present study, we investigate whether males of L. distinguendus use
chemical cues from larval feces of S. granarius for chemical orientation, as
females do. Furthermore, volatile constituents of the feces are identified, and
individual synthetic compounds are tested for biological activity. Finally, we
show that the volatile composition of the feces is due to the host-associated
mite fauna.
METHODS AND MATERIALS
Insects. S. granarius were reared on 250 g of wheat grains of about 14%
moisture content in 2-liter glass jars at a constant temperature of 25 C and 75%
relative humidity. To obtain weevil larvae of known age, 30 ml of adult weevils
were kept on 300 ml grain and removed seven days later. To rear L. distinguen-
dus, newly emerged parasitoids were placed in Petri dishes with grains contain-
ing 15- to 30-day-old weevil larvae. To obtain experienced parasitoids, batches
of 30 individuals (20 females and 10 males, 1–24 hr old) of L. distinguen-
dus were allowed to mate and oviposit in Petri dishes containing 60 infested
kernels and 60 mg of feces infested with Tyrophagus putrescentiae. One hour
before the experiments, parasitoids were removed from the Petri dishes and kept
individually in 1.5-ml polyethylene reaction tubes (Sarstedt, Nurnbrecht, Ger-
¨
many). Unmated females were collected immediately after hatching and kept
isolated from males until the experiments. Tyrophagus putrescentiae mites were

MITES AND PARASITOID ATTRACTION
1207
reared in Petri dishes on larval feces from S. granarius at 25 C and 85% relative
humidity.
Preparation of Extracts. Larval feces from S. granarius were obtained by
sieving grain infested by 19- to 30-day-old weevil larvae. Hexane extracts were
prepared by treating 20 g feces for 15 min with 40 ml hexane (Biomol, Ham-
burg, Germany) at room temperature in an ultrasonic bath. After filtration, 500
ml of the extract were used in the bioassay. Headspace extracts were obtained
by use of the closed-loop stripping (CLS) technique (Brechbuhler AG, Zurich,
¨ ¨
Switzerland). For this purpose, the volatiles from 5 g feces were trapped for
4 hr at room temperature on the charcoal layer (5 mg) of the adsorption tube.
The volatiles were eluted twice with 25 ml dichloromethane each (Biomol). The
combined eluates were used in the bioassay. For quantification of the volatiles,
the adsorption tube was eluted twice with 25 ml dichloromethane containing
1-dodecene at a concentration of 10 ng ml as an internal standard. The com-
/
bined eluates were analyzed by GC-MS.
Mite extracts were obtained by rinsing 20 Tyrophagus putrescentiae mites
with 20 ml hexane for 3 min. After removal of the mites, the extract was used
for chemical analysis.
Synthetic Mixture of Mite Semiochemicals. Based on the quantitative
chemical analysis of the CLS extracts, a mixture of synthetic mite semiochemi-
cals was prepared containing tridecane (Aldrich, Steinheim, Germany) at 60
mg ml, citral (mixture of neral and geranial; Roth, Karlsruhe, Germany) at 10
/
mg ml, and neryl formate at 5 mg ml. Neryl formate was synthesized by esterifi-
/
/
cation of nerol (Aldrich) with formic acid (Aldrich) by use of 4-dimethylamino
pyridine as catalyst and dicyclohexyl carbodiimide as condensation reactant, as
has been described in detail elsewhere for similar esters (Ruther et al., 1998).
Bioassays. Experiments were performed in a static four-chamber olfac-
tometer (Figure 1) made of acrylic glass, consisting of a cylinder (4 cm high
× 19 cm diam.) divided by vertical plates into four chambers. In one chamber,
an odor sample was placed in a Petri dish (5.5 cm diam.) laid out with brown
filter paper (4 cm diam.). The remaining chambers contained Petri dishes with
filter paper as controls. A walking arena (19 cm diam. × 1 cm high) consist-
ing of plastic gauze (mesh 0.5 mm), a rim of acrylic glass, and covered with
a glass plate was placed on the cylinder. The olfactometer was placed on the
bottom of a white bucket (29 cm diam. × 36 cm high) and illuminated from
above resulting in 2050 lux in the olfactometer. To avoid biased results due to
side preferences of the parasitoids, the position of the samples and the controls
was changed clockwise after each test. To avoid contamination of the walking
arena with sample odors or by possible pheromones of the parasitoids, walking
arenas and glass plates were cleaned regularly with ethanol and dimineralized
water. The parasitoids were exposed to the following samples: (1) 10 unmated
females—in these experiments, the sample container was sealed by gauze (mesh

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RUTHER AND STEIDLE
FIG. 1. Static four-chamber-olfactometer used for bioassays. For details see text.
0.5 mm), and thus, semiochemicals were able to diffuse to the walking arena
and the females were prevented from escaping, (2) 150 mg larval feces from S.
granarius; (3) 500 ml hexane extract from larval feces; (4) 50 ml CLS extract
from larval feces; and (5) 50 ml of the synthetic mixture of mite semiochemicals.
Samples 1–4 were tested with male parasitoids exclusively, sample 5 was tested
with both sexes.
When extracts were tested, the control chambers contained filter papers
treated with the pure solvents. Before the bioassays, the solvents were allowed
to evaporate for 15 min. For all experiments, samples were renewed after five
parasitoids each. The parasitoids were released individually in the center of the
walking arena and their behavior (walking, resting) and position in the four sec-
tors above the chambers were registered for 10 min by using the computer soft-
ware The Observer 3.0 (Noldus, Wageningen, The Netherlands, 1989). When
testing L. distinguendus in the described bioassay, about 20% of the parasitoids
rested more than 50% of the observation time. Those parasitoids were assumed to
be unmotivated and, therefore, were not included in the statistical analysis. The
time the parasitoids spent within the four sectors was analyzed for homogene-
ity by using the repeated measures ANOVA for dependent data (Bortz, 1993).
In case of significant deviation from homogeneity, the Scheffe-test for multiple
´
comparisons was used. In cases when data were not distributed equally, statis-
tical analysis was done by a nonparametric Friedman test, followed by Wilcox-
Wilcoxon tests for separation of means. Statistical analyses were done by using
Statistica scientific software (StatSoft, Hamburg, Germany).

MITES AND PARASITOID ATTRACTION
1209
GC-MS Analysis. Analytical separations were performed on a Fisons 8060
GC. Mass spectra were obtained on a Fisons MD800 quadrupole mass spectrom-
eter (Thermoquest, Egelsbach, Germany). Analyses were carried out by using
a 30-m × 0.32-mm-ID DB-5ms fused silica column, film thickness 0.25 mm
(J&W Fisher Scientific, Wiesbaden, Germany), with helium as carrier gas (pres-
/
sure 10 kPa). The temperature program started at 40 C, temperature was held
for 1 min, and raised 4 C min to 280 C. The column effluent was ionized by
/
electron impact ionization (EI) at 70 eV. Injection volumes were 1 ml (splitless)
in the case of the CLS extracts and 2 ml (splitless) in the case of mite extracts.
Kovats indices were estimated according to van den Dool and Kratz (1963) by
coinjection of a hydrocarbon mixture (C7–C30; Aldrich). Location of the double
bonds in the alkenes occurring in the extracts was determined by iodine cat-
alyzed methylthiolation that used dimethyl disulfide (Aldrich) and GC-MS anal-
ysis of the formed derivatives (Francis and Veland, 1981; Howard et al., 1988).
Quantitative analysis (N 2) of the volatiles in the CLS extracts was done by
comparing the peak areas of individual compounds with the peak area resulting
from the coinjection of 10 ng of the internal standard (1-dodecene).
RESULTS
Bioassays. When L. distinguendus males were exposed to 10 unmated
females in the olfactometer, they did not prefer the test sector over the control
sectors (Figure 2). On the other hand, they strongly preferred the test sector when
larval feces from S. granarius were presented. The chemicals responsible for
this behavior are extractable with hexane, as extracts from larval feces elicited
strong preference of the test sector by males. Selective enrichment of volatile
compounds from larval feces by use of CLS led to highly active extracts as well.
A synthetic mixture containing neral, geranial, neryl formate, and
tridecane—compounds occurring in both CLS extracts from larval feces and
astigmatid mites living in the feces (see below)—was tested towards males and
females of L. distinguendus. Both sexes preferred the test sector over the control
sectors when the synthetic mixture was presented in the olfactometer (Figure 3).
Astigmatid Mites. The behaviorally active larval feces from S. granarius
contained a high number of astigmatid mites, with one acarid species predomi-
nating, the mold mite, Tyrophagus putrescentiae (Schrank).
Chemical Analyses. Ten compounds were identified by GC-MS in the
behaviorally active CLS extracts from larval feces of S. granarius (Table 1):
four terpenoids, five hydrocarbons, and one aromatic compound. The identity
of another aromatic is still unknown. The amounts per 50 ml, i.e., the volume
used in the bioassay, were between 50 ng for geranial and 3000 ng for tridecane
(Table 1). All compounds identified in the CLS extracts also occurred in hex-

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RUTHER AND STEIDLE
FIG. 2. Mean allocation times ( SD, N 20) of L. distinguendus males in the sectors
of a four-chamber-olfactometer (T test, C1–C3 controls). The test field contained
10 unmated females (a), larval feces from the host S. granarius (b), hexane extracts
from feces (c), or extracts from CLS of the feces (d). Means with different letters are
significantly different at P < 0.001 (a,d: repeated measures ANOVA for dependent
data Scheffe
´
-test for multiple comparisons; b,c: Friedman test Wilcox-Wilcoxon test).
/
/
ane extracts from 20 T. putrescentiae mites (Figure 4). Thus, the composition
of volatiles in the larval feces from S. granarius was essentially determined by
coexisting astigmatid mites.
DISCUSSION
L. distinguendus males showed no preferences when 10 receptive females
were presented in a static four-chamber olfactometer. As we did not find any
evidence for the existence of a female-produced attractant, we expect that males
have to use other volatile sources for female location. Females of L. distinguen-
dus are strongly attracted to volatiles emitted by larval feces of their host, the
¨
granary weevil S. granarius (Steidle and Scholler, 1997). This study shows that
males are using the same host related cues for chemical orientation as females.

MITES AND PARASITOID ATTRACTION
1211
FIG. 3. Mean allocation times ( SD, N 25) of L. distinguendus females and males in
the sectors of a four-chamber-olfactometer (T test, C1–C3 controls). The test field
contained a mixture of synthetic chemicals occurring in extracts from feces of S. gra-
narius (see text for concentration). Means with different letters are significantly different
at P < 0.05 (repeated measures ANOVA for dependent data Scheffe-test for multiple
´
/
comparisons).
Females of L. distinguendus are receptive at the moment of emergence. Thus,
males that rely on the same chemical cues increase the probability of mating not
only with host-locating females but also with hatching females at the oviposition
sites. Chemical analyses of CLS extracts from larval feces of S. granarius led
TABLE 1. COMPOUNDS IN EXTRACTS FROM CLOSED-LOOP STRIPPING (CLS) OF LARVAL
FECES FROM GRANARY WEEVIL, S. granariusa
Concentration in
the CLS extract
No.
Compound
(ng 50 ml)
Identification
/
1
2
3
4
5
6
7
8
9
2-hydroxy-6-methylbenzaldehyde
neral
geranial
neryl formate
unknown (mw 150)
tridecane
200
450
50
Leal et al. (1988)
MS RI
/
MS RI
/
250
200
3000
150
620
350
630
130
MS RI
/
MS RI
/
b-acaridial
Leal et al. (1989)
DMDS
(Z,Z)-6,9-pentadecadiene
(Z)-7-pentadecene
(Z)-6-pentadecene
pentadecane
DMDS
DMDS
10
11
MS RI
/
a
Identification of the compounds is based on comparison of mass spectra and linear retention indices
with those of authentic reference compounds (MS RI), on interpretation of the mass spectra of the
/
reaction products after derivatization with dimethyl disulfide (DMDS), or on comparison of mass
spectra with data from the literature.

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RUTHER AND STEIDLE
FIG. 4. Total ion current chromatograms of a CLS extract from larval feces of S. gra-
narius (a) and a hexane extract of T. putrescentiae mites (b). The numbers correspond to
those in Table 1, is internal standard.

MITES AND PARASITOID ATTRACTION
1213
to the identification of 10 main compounds. A synthetic mixture containing four
of these constituents, neral (2), geranial (3), neryl formate (4), and tridecane (6)
attracted both sexes of L. distinguendus (Figure 4).
An interesting aspect of this study is the fact that the biologically active
compounds do not originate from the host feces themselves but from astigmatid
mites living in the feces. In the system investigated, all main compounds found
in the CLS extracts from the feces are due to the predominating mite species,
the mold mite T. putrescentiae. Numerous species of astigmatid mites have been
shown to communicate via pheromones. The compounds involved act as alarm,
sex, or aggregation pheromones (Kuwahara, 1991). T. putrescentiae has been
intensively investigated with respect to its chemical ecology. Neral (2), gera-
nial (3), and neryl formate (4) have been identified as alarm pheromones in
this species (Kuwahara et al., 1975, 1979; My-Yen et al., 1980). These com-
pounds and tridecane (6) were selected for bioassays because they not only occur
in T. putrescentiae but are widespread among mites infesting stored products.
Neral and geranial have been found in at least 14 species and, neryl formate in
9 (Kuwahara, 1991). Tridecane is also a common compound found in solvent
extracts from many astigmatid mites, often occurring as the major compound
(Curtis et al., 1981; Howard et al., 1988; Tuma et al., 1990). The function of this
compound is still unknown. Possibly it serves as a solvent for the actual semio-
chemicals. Tridecane has been suggested as an indicator substance to monitor
mite infestation in stored grain (Tuma et al., 1990).
2-Hydroxy-6-methylbenzaldehyde (1) is a well-known constituent of sev-
eral astigmatid mite species, among them T. putrescentiae (Kuwahara, 1991).
However, biological functions have only been shown for other species. It has
been described as an alarm pheromone in T. perniciosus (Leal et al., 1988),
whereas in Acarus immobilis and Aleuroglyphus ovatus, it mediates sexual
behavior (Kuwahara, 1991; Kuwahara et al., 1992). The terpenoid dialdehyde
b-acaridial (7) was identified for the first time in T. putrescentiae (Leal et al.,
1989), and again, biological activity has been shown only for another species,
the acarid mite Caloglyphus polyphyllae (Leal and Kuwahara, 1989). The C15
hydrocarbons identified in this study have been described previously from T.
putrescentiae and T. neiswanderi (Howard et al., 1988). In the latter species,
(Z)-7-pentadecene (9) and (Z)-6-pentadecene (10) have been shown to be com-
ponents of the alarm pheromone (Kuwahara et al., 1989).
To our knowledge, this study is the first to demonstrate that both sexes of a
parasitoid respond to pheromones of a host-associated nonmicroorganism. Until
now only host-associated microorganisms have been shown to produce volatile
metabolites that attract parasitoids. Thibout et al. (1993) found that volatile di-
alkyl disulfides attracting the parasitoid Diadromus pulchellus are produced by
bacteria living in the feces of the host, Acrolepiopsis assectella. Greany et al.
(1977) have shown that females of the fruit fly parasitoid Biosteres longicaudatus

1214
RUTHER AND STEIDLE
FIG. 5. Possible scenario for mate and host finding of L. distinguendus in the system
wheat (Triticum aestivum) S. granarius T. putrescentiae (for details see text).
/
/
are attracted by fermentation products of host associated fungi such as ethanol,
acetaldehyde, and acetic acid. Madden et al. (1968) investigated host finding in
Ibalia leucospoides, a parasitoid of the woodwasp Sirex noctilio, and found that
female parasitoids respond to the volatiles emitted by the fungus Amylostereum
sp. This fungus is symbiotically associated with the host and is inoculated into
coniferous trees during oviposition by Sirex females.
From the results of the present paper, a fascinating scenario of host and
mate finding in L. distinguendus can be deduced (Figure 5). Intense infestation
of stored wheat by the primary pest S. granarius causes mechanical digestion
of the substrate and an increase of moisture and temperature, resulting in so-
called hot spots (Sinha and Wallace, 1966). These conditions favor secondary
infestation by moisture-sensitive astigmatid mites that are often associated with
beetle-induced hot spots (Sinha, 1961; Eighme, 1966). Consequently, volatiles
emitted by these mites should reliably indicate the presence of potential hosts.
Thus, both sexes of L. distinguendus responding to these volatiles benefit from
this behavior, as it facilitates the location of areas of high host density as well as
mates. The fact that L. distinguendus responded to compounds that are common
for many stored-product-infesting mite species increases the ecological signifi-
cance of the observed phenomenon. Categorization of the bioactive chemicals as
kairomones appears equivocal at first glance, as the emitters of the compounds,
i.e., the mites, are not the actual target of the parasitoids. However, as beetle

MITES AND PARASITOID ATTRACTION
1215
infestation improves the living conditions for astigmatid mites in stored grain,
we conclude that attraction of beetle parasitoids causes disadvantages for the
mites as well and, thus, mite volatiles can be categorized as kairomones.
As mentioned before, the compounds tested in this study are known to act
as alarm pheromones in astigmatid mites. The investigated system not only con-
tained astigmatid mites but also predatory mites as possible antagonists. There-
fore, these organisms might be involved in the observed phenomena as elicitors
of the alarm pheromone emission in the astigmatid mites. This aspect needs fur-
ther investigation.
The parasitoids tested were experienced. Like numerous other insects, L.
distinguendus has been shown to learn from experience (Steidle, 1998). Thus,
the observed behavior may be due to associative learning. This aspect is currently
under investigation.
The synthetic mixtures of volatiles tested in this study did not elicit the same
degree of attractiveness towards L. distinguendus as extracts from larval feces.
This might be due to other compounds that have not yet been tested. However,
since L. distinguendus should also be able to locate hosts and mates in those
habitats where secondary infestation by astigmatid mites has not yet occurred,
there may be other chemicals that are involved in host and mate finding in this
species.
Acknowledgments—The authors thank Prof. A. Attygalle and K. B. Herath for confirming the
configuration of the double bonds of the alkenes, Dr. C. Corente for determining the Tyrophagus
¨
mites, and Silvia Nabe and Jana Wolfram for conducting some of the bioassays, Dr. Felix Wackers
and two anonymous reviewers made useful comments on an earlier draft of this manuscript.
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