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Vienna International Centre, PO Box 100 1400 Vienna, Austria Phone: (+43 1) 2600 - Fax: (+43 1) 26007 Email: [email protected],
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).