Research Article |
Corresponding author: Lucie Vaníčková ( luci.vanickova@gmail.com ) Academic editor: Teresa Vera
© 2015 Lucie Vaníčková, Radka Břízová, Antonio Pompeiano, Luana Lima Ferreira, Nathaly Costa de Aquino, Raphael de Farias Tavares, Laura D. Rodriguez, Adriana de Lima Mendonça, Nelson A. Canal, Ruth Rufino Do Nascimento.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Vaníčková L, Břízová R, Pompeiano A, Ferreira LL, de Aquino NC, Tavares RF, Rodriguez LD, Mendonça AL, Canal NA, do Nascimento RR (2015) Characterisation of the chemical profiles of Brazilian and Andean morphotypes belonging to the Anastrepha fraterculus complex (Diptera, Tephritidae). In: De Meyer M, Clarke AR, Vera MT, Hendrichs J (Eds) Resolution of Cryptic Species Complexes of Tephritid Pests to Enhance SIT Application and Facilitate International Trade. ZooKeys 540: 193-209. https://doi.org/10.3897/zookeys.540.9649
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Fruit fly sexual behaviour is directly influenced by chemical and non-chemical cues that play important roles in reproductive isolation. The chemical profiles of pheromones and cuticular hydrocarbons (CHs) of eight fruit fly populations of the Andean, Brazilian-1 and Brazilian-3 morphotypes of the Anastrepha fraterculus cryptic species complex originating from Colombia (four populations) and Brazil (four populations) were analysed using two-dimensional gas chromatography with mass spectrometric detection. The resulting chemical diversity data were studied using principal component analyses. Andean morphotypes could be discriminated from the Brazilian-1 and Brazilian-3 morphotypes by means of male-borne pheromones and/or male and female CH profiles. The Brazilian-1 and Brazilian-3 morphotypes were found to be monophyletic. The use of chemical profiles as species- and sex-specific signatures for cryptic species separations is discussed.
Cryptic species, chemotaxonomy, GC×GC/MS, PCA
Communication is a crucial process for both intra- and interspecific interactions (
The South American fruit fly, Anastrepha fraterculus (Wiedemann 1830) (Diptera: Tephritidae) constitutes a cryptic species complex with different degrees of pre- and postzygotic isolation (reviewed by
It is thought that tephritid fruit flies use contact pheromones in the last phase of courtship when a female briefly touches a male with its proboscis or front legs (
In A. fraterculus age- and sex-dependent CHs production has been investigated in one Argentinean population (
The present work aims to (i) clarify differences in the composition of male-borne volatiles among eight different populations belonging to three morphotypes (Andean, Brazilian-1 and Brazilian-3) of the A. fraterculus cryptic species complex; (ii) evaluate the potential use of male CH profiles for A. fraterculus identification of the Andean, Brazilian-1 and 3 morphotypes; (iii) investigate divergence in female CH profiles of the Andean and Brazilian morphotypes.
Eight laboratory populations, previously analysed for the morphotype identification by
Geographical location of the sampled populations of Anastrepha fraterculus in Brazil and Colombia and the analyses of their cuticular hydrocarbons (CH) and male-borne pheromones.
Country, State | Population | Code | Alt. [m] | Latitude | Longitude | Morphotype |
CH analyses | Pheromone analyses |
---|---|---|---|---|---|---|---|---|
Brazil, RS | Pelotas |
PEL | 7 | 29°28.19'S | 50°37.03'W | Brazilian-1 | + | + |
Brazil, RS | Bento Gonçalves |
BEN | 690 | 29°17.08'S | 51°51.89'W | Brazilian-1 | + | + |
Brazil, SC | São Joaquim |
SAO | 1360 | 28°17.38'S | 49°55.54'W | Brazilian-1 | + | + |
Brazil, AL | Alagoas |
AL | 16 | 10°08.01'S | 36°10.34'W | Brazilian-3 | + | + |
Colombia | Ibague |
IBA | 1580 | 04°26.20'N | 75°13.55'W | Andean | n.a. | + |
Colombia | Sibundoy |
SIB | 2136 | 01°20.33'N | 76°91.92'W | Andean | + | + |
Colombia | Duitama |
DUI | 2569 | 05°49.50'N | 73°04.49'W | Andean | + | + |
Colombia | Cachipay |
CAC | 1850 | 04°45.27'N | 74°23.02'W | Andean | + | n.a. |
For the male-volatile collection following populations were available: three Colombian (Duitama, Ibague, Sibundoy) and four Brazilian (Alagoas, Bento Gonçalves, Pelotas, São Joaquim) (Table
For the extraction of CHs following populations were available: three Colombian (Duitama, Cachipay, Sibundoy) and four Brazilian (Alagoas, Bento Gonçalves, Pelotas, São Joaquim) (Table
Two-dimensional gas chromatography with time-of-flight mass spectrometric detection (GC×GC/MS) was used for the quantification and identification of male-borne volatiles and CH profiles. Identical conditions were used for the analyses of all samples and for chromatographic data evaluation, as described in previous studies on A. fraterculus male pheromones (
Except for (S,S)-(-)-epianastrephin, which was provided by Prof. Jim Nation (University of Florida, Gainesville, USA), all chemicals were purchased either from Sigma-Aldrich, Brazil [α-pinene, limonene, (Z)-3-nonen-1-ol] or from Penta, USA [(E, Z)-3,6-nonadien-1-ol]. Chemicals were > 95% pure, based on the results from capillary gas chromatography.
The relative peak areas of seven male-borne volatile compounds and forty-eight male and female CHs (as identified by the GC×GC/MS in the deconvoluted total-ion chromatogram mode) were calculated for each replicate of the study populations.
The differences in the chemical composition of the samples from study populations were analysed by principal component analysis (PCA). Prior to PCA, peak areas were subjected to logarithmic transformation; intraspecific scaling was performed by dividing each species score by its standard deviation; the data were centred by species’ scores. In PCA analyses, hierarchical clustering based on Pearson correlation showed that populations with similar chemical profiles cluster together.
A heat map was used to visualize male-borne volatiles organized as matrices. The heat map performed two actions on a matrix. First, it reordered the rows and columns so that rows and columns with similar profiles were closer to one another, causing these profiles to be more visible to the eye. Second, each entry in the data matrix was displayed as a colour, making it possible to view the patterns graphically. Dendrograms were created using correlation-based distances and the Ward method of agglomeration was used in the present analysis (
Significant quantitative differences (P < 0.05) were found in the male-borne volatiles, namely α-pinene, limonene, (Z)-3-nonen-1-ol, (E, Z)-3,6-nonadien-1-ol, (Z, E)-α-farnesene, (E, E)-α-farnesene and epianastrephin, among the investigated populations of A. fraterculus. A heat map was constructed to visualize the relative proportions of the seven volatiles in each of the populations (Figure
Heat map of seven male-borne volatiles (columns) identified by GC×GC/MS analyses in seven populations (rows) of the Anastrepha fraterculus cryptic species complex. The dendrograms were created using correlation-based distances and the Ward method of hierarchical clustering (P < 0.05). Key: AL – Alagoas, AL, Brazil; BEN – Bento Gonçalves, RS, Brazil; DUI – Duitama, Colombia; IBA – Ibague, Colombia; PEL – Pelotas, RS, Brazil; SAO – São Joaquim, SC, Brazil; SIB – Sibundoy, Colombia. Epianas – Epianastrephin; Z.E.Far – (
The PCA analyses of GC×GC/MS data showed the Andean morphotype formed one cluster while Brazilian-1 and Brazilian-3 morphotypes formed another cluster (Figure
Principal component analyses (PCA) of transformed GC×GC/MS data of seven male-borne volatiles produced by groups of 20 sexually mature individuals from seven populations of the Anastrepha fraterculus cryptic species complex. Variables factor map represents projection of variables on the plane defined by the first two principal components. Hierarchical clustering is score plot describing the populations and their clustering. Key: AL – Alagoas, AL, Brazil; BEN – Bento Gonçalves, RS, Brazil; DUI – Duitama, Colombia; IBA – Ibague, Colombia; PEL – Pelotas, RS, Brazil; SAO – São Joaquim, SC, Brazil; SIB – Sibundoy, Colombia. Epianas. – Epianastrephin; Z.E.Far – (
Forty-eight male CHs, including 19 linear n-alkanes (A1-19), 11 methylbranched alkanes (B1-11), 11 alkenes (C1-11), and 7 alkadienes (D1-7), were evaluated by PCA for the possible use in the identification of Andean (CAC, DUI, SIB), Brazilian-1 (BEN, PEL, SAO) and Brazilian-3 (AL) morphotypes (Figure
Principal component analyses (PCA) of transformed GC×GC/MS data of 48 male CHs from seven populations of the Anastrepha fraterculus cryptic species complex. Variables factor map represents projection of variables on the plane defined by the first two principal components. Hierarchical clustering is score plot describing the populations and their clustering. Key: AL – Alagoas, AL, Brazil; BEN – Bento Gonçalves, RS, Brazil; CAC – Cachipay, Colombia; DUI – Duitama, Colombia; PEL – Pelotas, RS, Brazil; SAO – São Joaquim, SC, Brazil; SIB – Sibundoy, Colombia. A1–19 –
Female CH profiles from study populations consisted of 48 saturated and unsaturated compounds with chain lengths ranging from 12-38 carbons. In female body washes, unsaturated male-specific CHs were absent, namely 7-heneicosene, 7-docosene, 7-tricosene and 7-pentacosene. In the PCA analyses the populations segregated into two main groups (Figure
Principal component analyses (PCA) of transformed GC×GC/MS data of 48 female CHs from seven populations of the Anastrepha fraterculus cryptic species complex. (Variables factor map) projection of variables on the plane defined by the first two principal components. (Hierarchical clustering) score plot describing the populations and their clustering. Key: AL – Alagoas, AL, Brazil; BEN – Bento Gonçalves, RS, Brazil; CAC – Cachipay, Colombia; DUI – Duitama, Colombia; PEL – Pelotas, RS, Brazil; SAO – São Joaquim, SC, Brazil; SIB – Sibundoy, Colombia. A1–19 –
Chemical profiles of A. fraterculus varied quantitatively among populations from diverse regions of South America. To some extent, the chemical profiles showed comparable patterns among populations belonging to the same morphotype. Comparison of the chemical profiles of the Andean and two Brazilian morphotypes showed that the more geographically distant the morphotypes are, the more diverse their pheromone and CH profiles are. Nevertheless, this trend was not observed between the Brazilian-1 and Brazilian-3 morphotypes. Within the A. fraterculus complex, the Andean morphotype is allopatric, while the Brazilian morphotypes (Brazilin-1, Brazilian-2 and Brazilian-3) are sympatric (
The Andean morphotype populations had very different pheromone profiles from that of the two Brazilian morphotypes.
Male and female flies of the Andean morphotype and the Brazilian morphotypes can be separated using CH profiles (
CHs in insects serve primarily to prevent desiccation by reducing water loss (
The present study demonstrates that pheromone components and CH profiles diverge qualitatively between Andean and Brazilian-1 and Brazilian-3 morphotypes and may be used to some extent to delimit morphotypes in the A. fraterculus species complex. Comprehensive studies, which simultaneously examine environmental, behavioural, genetic and chemical features are necessary to be performed aiming to understand which factors affect the geographical variation in the male-borne volatiles and CH profiles in the A. fraterculus complex.
The authors acknowledge Dr. Tara Massad for the language corrections and valuable comments, and the anonymous reviewers for their comments on the early version of the manuscript. Financial support was provided by the Institute of Organic Chemistry and Biochemistry, Academy of Science of Czech Republic, Prague (RVO: 61388963) and the Conselho Nacional de Desenvolvimento Científico e Tecnologico, Brazil (CNPq 376348/2012-7). The authors sincerely acknowledge support provided by the FAO/IAEA via the Coordinated Research Project ‘Resolution of cryptic species complexes of tephritid pests to overcome constraints to SIT application and international trade’.
Table S1. Anastrepha fraterculus male and female characteristic.
Data type: species data
Explanation note: Anastrepha fraterculus male (m) and female (f) characteristic cuticular hydrocarbons identified by principal component analyses.