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Vienna International Centre, PO Box 100 1400 Vienna, Austria Phone: (+43 1) 2600 - Fax: (+43 1) 26007 Email: Official.Mail@iaea.org,

PROGRESS REPORT OF RESEARCH CONTRACT No: 16106

CONTENT

TASKS PLANNED FOR 2012 PAGE 1

TASKS ACCOMPLISHED IN 2012 – SUMMARY PAGE 2

TASKS ACCOMPLISHED IN 2012 – DETAILS PAGE 3

NOT FINISHED TASKS PAGE 13

PLANS FOR 2013 PAGE 14

TASKS PLANNED FOR 2012

In the year 2012 we planned to:

Finish the following publications:

o Larval food influences the composition of male sex pheromone in Ceratitis capitata

o Cuticular hydrocarbons of the South American fruit fly Anastrepha fraterculus: Variability

with sex and age

Finish data evaluation of CHC analysis of the two genetically modified populations C. capitata

Finish data evaluation of CHC analysis of the two Brazilian reproductively isolated populations of A.

fraterculus

Finish data evaluation of CHC distribution on A. fraterculus body surface by means of MALDI

imaging analysis

Finish data evaluation of CHC analysis of Ceratitis FAR complex

Continue investigate Ceratitis FAR complex pheromone, extend the study for C. rosa and C.

fasciventris subpopulations, if possible

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TASKS ACCOMPLISHED IN 2012 - SUMMARY

Published papers

Vaníčková L., do Nascimento R.R., Hoskovec M., Ježková Z., Břízová R., Tomčala A., Kalinová B.:

Are the Wild and Laboratory Insect Populations Different in Semiochemical Emission? The Case of

the Medfly Sex Pheromone. Journal od Agricultural and Food Chemistry 60 (29): 7168-7176 (2012).

Vaníčková L., Svatoš A., Kroiss J., Kaltenpoth M., do Nascimento R.R., Hoskovec M., Břízová R.,

Kalinová B.: Cuticular Hydrocarbons of the South American Fruit Fly Anastrepha fraterculus:

Variability with sex and age. Journal of Chemical Ecology 38 (9): 1133-1142 (2012).

Participation on conferences

Vaníčková, L.; Břízová, R., Tomčala, A.; Hoskovec, M.; Kalinová, B.: Anastrepha complex and

Ceratitis FAR complex (Diptera: Tephritidae). In 2nd

CRP meeting: Resolution of cryptic species

complexes of Tephritid pests to overcome constraints to SIT application and international trade,

Brisbane, Australia 30.1-3.2.2012.

Vaníčková, L.; Virgilio, M.; Břízová, R.; Hoskovec, M.; Kalinová, B.: Cuticular hydrocarbons as a

diagnostic tool for the identification of species within the Ceratitis FAR complex (Diptera:

Tephritidae). Book of abstracts p. 68. In 2nd

International Symposium of TEAM. Kolymbari, Greece 3-

6.7. 2012.

Břízová, R.; Vaníčková, L., Kalinová, B.; Faťarová, M.; Hoskovec, M.: Pheromonal communication

in Ceratitis FAR complex (Diptera: Tephritidae). Book of abstracts p. 64. In 2nd

International

Symposium of TEAM. Kolymbari, Greece 3-6.7. 2012.

Břízová, R.; Vaníčková, L., Kalinová, B.; Faťarová, M.; Hoskovec, M.: Chemotaxonomical

relationships within the Ceratitis FAR complex (C. fasciventris, C. anonae and C. rosa): I. based on

the pheromone analysis. Book of abstracts p. 148. In 28th

ISCE Annual Meeting; Vilnius, Lithuania

22.-26.7. 2012.

Břízová, R.; Vaníčková, L., Kalinová, B.; Faťarová, M.; Hoskovec, M.: Chemotaxonomical

relationships within the Ceratitis FAR complex (C. fasciventris, C. anonae and C. rosa): II. based on

the cuticular hydrocarbons analysis. Book of abstracts p. 176. In 28th

ISCE Annual Meeting; Vilnius,

Lithuania 22.-26.7. 2012.

Ph.D. and M.Sc. theses

Lucie Vaníčková, M.Sc. - Ph.D. thesis: Chemical ecology of fruit flies genera Anastrepha and

Ceratitis, Institute of Chemical Technology, Prague, September 2012.

Zuzana Ježková - Analysis of male sex pheromone of tropic fruit fly Ceratitis capitata (Tephritidae),

M.Sc. thesis, Faculty of Sciences, Charles University, Prague, June 2012

Research

The data analysis of CHCs of Ceratitis FAR complex has been finished

The pheromone analysis of Ceratitis FAR complex has been finished; data are ready for publication

(presented below).

CHCs and male sex pheromones have been collected for 5 populations and 5 morphotypes. Data are

partially analyzed (details presented below).

CHCs distribution on A. fraterculus body surface by means of MALDI imaging analysis has been

attempted, preliminary results are presented bellow.

The age-related changes in the composition of male sex pheromone of A. fraterculus laboratory

population were analyzed. Data evaluation is in the progress.

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TASKS ACCOMPLISHED IN 2012 - DETAILS

CHC distribution on A. fraterculus body surface by means of MALDI imaging analysis

Our data show that CHC distribution on A. fraterculus surface is not uniform, but areas with specific CHC

accumulation exist (Fig. 1). These areas may play specific role in premating chemical communication. The

phenomenon will be further investigated (Vaníčková).

.

Figure 1: Male and female of A. fraterculus on the MALDI plate.

The color spots on the wings are polar compounds visualized by MALDI

imaging technique.

CHC and pheromone analysis of Ceratitis FAR complex

Chemical analyses (GC×GC-TOF/MS) of Ceratitis FAR complex revealed 118 cuticular hydrocarbons

(CHCs) present in all three species. The CHCs profiles are complex mixtures of straight-chained, methyl-

branched saturated and unsaturated hydrocarbons with a wide range of carbon backbones (C23–C38). In

comparison with previous reports, our analyses revealed a higher number of n-alkanes, short-chain branched

alkanes, long-chain alkenes, and alkadienes (Vaníčková). Representative GC×GC-TOF/MS chromatograms

of CHCs of the three species are depicted in Fig. 2. The identification of the compounds was based on the

mass spectra and retention indices (RI). To compare the composition between the three investigated species,

Principal Component Analysis (PCA) followed by Redundancy Analysis (RDA) (Biometris, Plant Research

International, Wageningen, Netherlands) were used.

Figure 2 depicts the GC×GC-TOF/MS analysis of cuticular hydrocarbons of males from Ceratitis FAR

complex. Each spot represents one compound. The numbers are retention indices of the most statistically

significant compounds that characterize the respective species.

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Figure 2:

In addition to species-specific also gender-specific differences in CHCs profiles were found Fig. 3 and 4. The

PCA (Fig. 3) shows a clear separation of the three species of the Ceratitis FAR complex. The two principal

components (PC1 and PC2) together accounted for 90% of the total variability. The subsequent RDA

confirmed significant differences in the chemical profiles of CHCs between each pair of the three species (P

= 0.001). The contribution of particular compounds to overall differences is depicted in Fig. 4 (Vaníčková).

Figure 3: The results of the multivariate principal

component analysis (PCA) of 118 nonpolar

compounds extracted from 10 males (circle) and 10

females (square) of C. anonae, C. rosa and C.

fasciventris. The 3 species are clearly segregated.

Figure 4: The results of the multivariate redundant

analysis (RDA) of the 118 nonpolar compounds

extracted from 10 males (circle) and 10 females

(square) of C. anonae, C. rosa and C. fasciventris.

The arrows represent 8 statistically significant

compounds that characterize the respective species.

C. anonae (red color), C. rosa (blue color) and C. fasciventris (green color).

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Sex-specific CHCs:

Both PCA and RDA showed significant differences between the sexes (P = 0.001) in all three species. An

example of the 2D chromatograms of male and female C. anonae is presented in (Fig. 5; Vaníčková). The

female specific CHCs are characterized by RI 2761, 2860, 3164, 3224, 3254, 3355, and 3476, male specific

CHC is characterized by RI 2983 In C. fasciventris, female specific CHC was 3435, male specific CHCs

were at RI 3093, 3224, 3284, 3294, 3365, 3489, 3495, and 3768. Ceratitis rosa males had sex-specific

hydrocarbons with following RI: 2943, 3013, 3123, 3526, 3536, and 3566. A female specific CHC was

identified at RI 3587.

Figure 5: An example of GC×GC-TOF/MS chromatograms of the most statistically significant sex-specific

nonpolar compounds from male and female C. anonae. The numbers express retention indices (Vaníčková).

Pheromones of FAR complex

Chemical analyses (GC×GC-TOF/MS) of pheromones of Ceratitis FAR complex revealed showed complex

mixtures of many compounds with different but overlapping profiles. The GC-EAD analyses showed

altogether 10 antenally active compounds (Tab. 1). In addition, 6-methyl-5-heptene-2-one and geranyl

acetone were found to elicited EAD responses in C. rosa, but these compounds were present also in blank, so

their origin is not clear. Tab. 1 shows that studied species share some active components but also have some

that are species-specific.

In conclusion, both CHCs and sex pheromones analyses show species-specific characteristics. The data

support the idea that species forming Ceratitis FAR complex employ species-specific chemical

communication and are reproductively isolated.

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Table 1:

Compounds RI (LECO) C. fasciventris C. anonae C. rosa

Methyl-(3Z)-hexenoate 937 +

Methyl-(2E)-2-hexenoate 966 +

6-Methyl-5-heptene-2-one* 989 *

α-myrcene 993 + +

Ethyl hexanoate 999 +

Ethyl (E)-3-hexenoate 1006 +

Linalool 1104 + + +

Methyl (3Z)-3-octenoate 1131 +

Nonen-2-enal 1163 + + +

Geranyl acetone* 1456 *

(E,E)-α-farnesene 1507 +

Methyl (2E,6E)-farnesoate 1799 + + +

* antennally active compounds, but present also in blank

Pheromones and CHCs in different A. fraterculus populations and morphotypes

Table 2: the overview of CHC analysis performed so far in A. fraterculus populations and morphotypes.

R12/IAEA – Radka Břízová collected samples in IAEA in 2012

R&L /IOCB – samples collected by Ruth Rufino do Nascimento or Lucie Vaníčková and analyzed in IOCB

LVP – Lucie Vaníčková postdoctoral fellowship

The preliminary data obtained from one- dimensional and two-dimensional gas-chromatographic / mass

spectrometric analyzes show differences in the CHCs profiles (Figures 5 - 9).

CHC 1 2 3 4 5 6 7 MRF Mexican Andean Venezuelan Peruvian

A. aff 1 A. aff 2 A. aff 3

Mexico

Colombia

Peru

Brazil

Brazil

Brazil

R12/IAEA

R12/IAEA

Not available Caceres

R12/IAEA

Tucuman

R12/ IAEA

R&L /IOCB

Bento-Goncalves

R&L /IOCB (LVP)

Vacaria

R/ IOCB R12/IAEA

(LVP)

Piracicaba

R12/IAEA (LVP)

Pelotas

missing (LVP)

Sao Joaquim

missing (LVP)

Alagoas

R&L /IOCB

(LVP)

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Figure 5

(A) Two-dimensional GC-MS CHCs analysis of Mexican morphotype

(B) One-dimensional GC-MS CHCs analysis of Mexican morphotype

Figure 6

(A) Two-dimensional GC-MS CHCs analysis of Andean morphotype

(B) One-dimensional GC-MS CHCs analysis of Andean morphotype

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Figure 7

(A) Two-dimensional GC-MS CHCs analysis of Peruvian morphotype

(B) One-dimensional GC-MS CHCs analysis of Peruvian morphotype

Figure 8

(A) Two-dimensional GC-MS CHCs analysis of Tucuman (A. aff 1 morphotype)

(B) One-dimensional GC-MS CHCs analysis of Tucuman (A. aff 1 morphotype)

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Figure 9

Two-dimensional GC-MS CHCs analysis of Alagoas population (A. aff 3 morphotype)

(B) One-dimensional GC-MS CHCs analysis of Alagoas population (A. aff 3 morphotype)

Evaluation of CHCs of remaining A. fraterculus population, e.g. Alagoas, São Joaquim, Pelotas, Bento

Gonçalves, Piracicaba, Vacaria will be performed during next period (in cooperation with Universidade

Federal de Alagoas, Maceio, Brazil - the location of postdoctoral fellowship of Dr. Lucie Vaníčková).

Pheromone analysis of A. fraterculus populations and morphotypes

The pheromone analyses performed so far are summarized in Table 3:

Table 3: Samples available for pheromone analysis

R12/IAEA – Radka Břízová collected samples in IAEA in 2012

R&L /IOCB – samples collected by Ruth Rufino do Nascimento or Lucie Vaníčková and analyzed in IOCB

PHER 1 2 3 4 5 6 7 MRF Mexican Andean Venezuelan Peruvian A. aff 1 A. aff 2 A. aff 3

Mexican Colombian Peru Brazilian Brazilian Brazilian

missing

missing

not available Caceres

R12/IAEA

Tucuman

R12/IOCHB

Bento-Goncalves

R&L /IOCB

Piracicaba

R12/IAEA

R&L/IOCB

Vacaria

R12/IAEA

R&L/IOCB

Pelotas

R&L/IOCB

Sao- Joaquim

R&L/IOCB

Alagoas

R&L/IOCB

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The GC-GC-MS and GC-EAD analysis of A. fraterculus pheromone revealed 10 antennally active

compounds (Progress Report 2012). The occurrence of these compounds in pheromone of 10 A. fraterculus

populations are depicted in Tab. 4 and Fig. 10. Both Tab. 4 and Fig. 10 show clear population specificity.

Table 4: The antennally active compounds identified by GC×GC/TOF-MS in the aeration extracts of calling

males of A. fraterculus from five Brazilian populations and four populations from Vienna.

No. Compound RI PIR PIR 12 VAC VAC 12 BEN PEL SJ TUC 12 PER 12

1 2-Ethylhexan-1-ol 1029 ++ + +++ + + tr + + +

2 (Z)-β-Ocimene 1050 + + tr + ++ + + ++ ++

3 (3Z)-Nonen-1-ol 1159 ++ ++++ tr ++++ tr tr tr +++ +++

4 (3E,6Z)-Nonadien-1-ol 1161 +++ +++ + ++ ++++ tr + ++ +++

5 (Z,E)-α-Farnesene 1495 + + tr + + + + + +

6 Germacrene D 1498 tr + ++ tr tr + tr tr tr

7 (E,E)-α-Suspensolide 1506 + + tr ++ + + + ++ +

8 (E,E)-α-Farnesene 1512 + ++ + +++ +++ + ++ +++ +++

9 Anastrephin 1617 + ++ + + + + + + +

10 Epianastrephin 1621 + ++ + ++ + + + ++ +

Piracicaba – PIR, Bento Gonçalves - BEN, Vacaria – VAC, Pelotas - PEL, São Joaquim – SJ and four

populations from Vienna: Piracicaba – PIR 12, Vacaria – VAC 12, Tucuman – TUC 12 and Peru - PER 12.

RI – retention indexes on DB-5 column.

Quantification: tr ≤ 0.3 %; + < 6 %; ++ ≤ 16 %; +++ < 30 %; ++++ > 35 %; +++++ > 40 %; ++++++ > 55 %.

Figure 10: Comparison of antennally active pheromone components released by A. fraterculus calling males

originated from five Brazilian populations and four populations from Vienna. Chromatographic peaks

indicate a hypothetical illustration of separation obtained based on the volatility of the compounds. Peak

heights represent relative amounts of the pheromone components.

The numbers represent respective compounds: (1029) 2-ethylhexan-1-ol, (1050) (Z)-β-ocimene,

(1159) (3Z)-nonen-1-ol, (1161) (3E,6Z)-nonadien-1-ol, (1495) (Z,E)-α-farnesene, (1498) germacrene D,

(1506) (E,E)-α-suspensolide, (1512) (E,E)-α-farnesene, (1617) anastrephin, (1621) epianastrephin.

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The multivariate principal component analysis (PCA) of the sex pheromone of the males of A. fraterculus

originating from five different populations (Bento Gonçalves – B01-05, Pelotas – PE01-05, Piracicaba – P01-

05, São Joaquim – S01-05,and Vacaria – V01-05) show clear separation (Fig. 11). As could be seen from the

figure 11, The Vacaria and Pelotas populations are distantly separated from each other and from the rest of

analyzed populations, e.g. from Bento Gonçalves, São Joaquim and Piracicaba populations, which are quite

closely related.

Figure 11: The results of the multivariate principal component analysis (PCA) of the sex pheromone of the

males of A. fraterculus originating from five different populations (Bento Gonçalves – B01-05, Pelotas –

PE01-05, Piracicaba – P01-05, São Joaquim – S01-05,and Vacaria – V01-05). The five populations are

clearly segregated. Each symbol on the plot represents one sample (blue – Bento Gonçalves population,

green – Vacaria population, red – São Joaquim population, yellow – Piracicaba population, rosa - Pelotas

population). The number of the analyzed samples (N) for each population was 5.

Pheromone analysis of five Brazilian populations of A. fraterculus (Bento Gonçalves, Piracicaba, Vacaria,

Pelotas, São Joaquim) are prepared for joint publication of Vanessa Simões Dias et al., Compatibilidade de

acasalamento de populações do complexo Anastrepha fraterculus (Diptera: Tephritidae) do Brasil. In

preparation. Pheromone analysis of populations Peru, Tucuman, Vacaria, Piracicaba already performed will

be finished within the framework of Radka Břízová Ph.D. study.

Variabilities of pheromone composition within laboratory populations originating from different

laboratories

We observed that differences in dynamic collection technique affects both qualitative and quantitative

composition of pheromone significantly. For instance, a comparison of pheromone composition of one

Piracicab A. fraterculus population from Brazil and Vienna showed qualitative difference in pheromone

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composition (Fig. 12). Aniother comparison for Vacaria A. fraterculus population from Brazil and Wienna

showed both qualitative and quantitative differences (Fig. 12,13) (Brazilian samples were obtained in Brazil

(Porapak). Vienna population was sampled in Vienna on Super Q with the technique available there. Both

Brazilian and Vienna samples were eluted by hexane and analyzed using GC×GC-MS/TOF technique in

IOCB. These data show that to depict real population differences, it is essential to compare pheromones

collected by the standardized technique, otherwise, the data may not be relevant. Another possibility is that

rearing conditions in Brazil and in Vienna led to population differenciation. However to validate this

standardized methods have to be used for pheromone collection.

Figure 12: Qualitative and quantitative differences of Brazilian and Austrian Piracicaba population of A.

fraterculus. The numbers represent respective compounds: (1029) 2-ethylhexan-1-ol, (1050) (Z)-β-ocimene,

(1159) (3Z)-nonen-1-ol, (1161) (3E,6Z)-nonadien-1-ol, (1495) (Z,E)-α-farnesene, (1498) germacrene D,

(1506) (E,E)-α-suspensolide, (1512) (E,E)-α-farnesene, (1617) anastrephin, (1621) epianastrephin.

Figure 13: Qualitative and quantitative differences of Brazilian and Austrian Vacaria population of A.

fraterculus. The numbers represent respective compounds: (1029) 2-ethylhexan-1-ol, (1050) (Z)-β-ocimene,

(1159) (3Z)-nonen-1-ol, (1161) (3E,6Z)-nonadien-1-ol, (1495) (Z,E)-α-farnesene, (1498) germacrene D,

(1506) (E,E)-α-suspensolide, (1512) (E,E)-α-farnesene, (1617) anastrephin, (1621) epianastrephin.

.

Our data related to age-dependent changes of pheromone show that young males do not produce sex

pheromone. The production starts several days after emergence and reaches peak at the age of maturity.

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During sexual maturity the pheromone blend remains relatively constant. The GC-EAD experiments show

that both sexes of A. fraterculus are equally able to perceive the pheromone and that this ability is acquired

immediately after emergence well before the flies reach the sexual maturation (Fig. 14). In conclusion, both CHCs and sex pheromones analyses show species-specific characteristics. The data support the

idea that some population of A. fraterculus complex employ species-specific chemical communication and because of

it they might evolved behavioral reproductive isolation.

Figure 14: Age-dependent antennal sensitivity of A. fraterculus males and females to male sex pheromone

In conclusion, components of A. fraterculus pheromnoes appear about 6 days after emergence and reach the peak at the

time of sexual maturity. On the other hand, the ability to perceive pheromone is established immediately after

emergence sex pheromones analyses show species-specific characteristics.

NOT FINISHED TASKS PLANNED FOR 2012

Analysis of CHCs and male sex pheromones of C. rosa and C. fasciventris subpopulations has not

been done yet, since the material is not available.

Analysis of CHCs and male sex pheromones of genetically modified population (masculinized

females) C. capitata has not been started yet, since the flies with genetically modified sex are now

being prepared.

Age dependent antennal sensitivity

0

50

100

150

0 1 2 5 8 12 16 20

Age (days)

EA

D (

mV

)

Males

Females

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PLANS FOR 2013

During the next research period we aim to

Analyse CHCs and pheromones of C. rosa and C. fasciventris sub-populations

Finish the analyses of CHC analysis of Vacaria, Piracicaba, and Colombia populations of A.

fraterculus complex

Complete the pheromone analysis of PERU, TUCUMAN, VACARIA, and PIRACICABA

populations of A. fraterculus complex

Collect and analyze the pheromone of the remaining A. fraterculus populations, e.g. Mexican,

Andean and Venezuelan morphotypes

Perform GC-EAD analysis of pheromones of distant A. fraterculus populations to see, whether

changes in pheromone composition are followed also in pheromone perception.

To perform CHC and pheromone analysis of sex manipulated C. capitata population to determine,

whether chemical communication of normal and masculinized XX females differ or not.

To finish the following publications:

o Pheromone analysis of five Brazilian populations of A. fraterculus (Bento Gonçalves,

Piracicaba, Vacaria, Pelotas, São Joaquim) are prepared for joint publication of Vanessa

Simões Dias et al., Compatibilidade de acasalamento de populações do complexo Anastrepha

fraterculus (Diptera: Tephritidae) do Brasil. In preparation.

o Vaníčková L., Tomčala A., Břízová R., Virgilio M., Do Nascimento R.R., Hoskovec M.,

Kalinová B., DeMeyer M. Is it possible to distinguish between cryptic species complexes

using cuticular hydrocarbons? African fruit flies complex on target.

o Břízová R., Vaníčková L., Faťarová M., Tomčala A., Hoskovec M., Kalinová B. Chemical

communication of Ceratitis FAR complex: pheromone and CHCs.

o R Břízová R, Vaníčková L Tomčala A, Hoskovec M, Do Nascimento R.R., Mendoca A.,

Kalinová B. CHC and pheromone analysis of A. fraterculus populations: Resolution of

Anastrepha fraterculus complex using pheromone and cuticular hydrocarbons.

o Břízová R., Vaníčková L., Tomčala A., Hoskovec M., Do Nascimento R.R., Mendoca A.,

Kalinová B. Age-dependent changes in pheromone production and perception in A.

fraterculus

REFERENCES

Lucie Vaníčková, M.Sc.: Ph.D. thesis: Chemical ecology of fruit flies genera Anastrepha and Ceratitis,

Institute of Chemical Technology, Prague, September 2012.

Vaníčková L., Svatoš A., Kroiss J., Kaltenpoth M., do Nascimento R.R., Hoskovec M., Břízová R., Kalinová

B.: Cuticular Hydrocarbons of the South American Fruit Fly Anastrepha fraterculus: Variability with sex and

age. Journal of Chemical Ecology 38 (9): 1133-1142 (2012).