Review Article |
Corresponding author: M. Teresa Vera ( teretina@hotmail.com ) Academic editor: Marc De Meyer
© 2015 María Laura Juárez, Francisco Devescovi, Radka Břízová, Guillermo Bachmann, Diego Fernando Segura, Blanka Kalinová, Patricia Fernández, María Josefina Ruiz, Jianquan Yang, Peter E.A. Teal, Carlos Caceres, Marc J.B. Vreysen, Jorge Hendrichs, M. Teresa Vera.
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:
Juárez ML, Devescovi F, Břízová R, Bachmann G, Segura DF, Kalinová B, Fernández P, Ruiz MJ, Yang J, Teal PEA, Cáceres C, Vreysen MJB, Hendrichs J, Vera MT (2015) Evaluating mating compatibility within fruit fly cryptic species complexes and the potential role of sex pheromones in pre-mating isolation. 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: 125-155. https://doi.org/10.3897/zookeys.540.6133
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The study of sexual behavior and the identification of the signals involved in mate recognition between con-specifics are key components that can shed some light, as part of an integrative taxonomic approach, in delimitating species within species complexes. In the Tephritidae family several species complexes have received particular attention as they include important agricultural pests such as the Ceratitis fasciventris (Bezzi), Ceratitis anonae (Graham) and Ceratitis rosa Karsch (FAR) complex, the Bactrocera dorsalis (Hendel) complex and the Anastrepha fraterculus (Wiedemann) complex. Here the value and usefulness of a methodology that uses walk-in field cages with host trees to assess, under semi-natural conditions, mating compatibility within these complexes is reviewed, and the same methodology to study the role of chemical communication in pre-mating isolation among A. fraterculus populations is used. Results showed that under the same experimental conditions it was possible to distinguish an entire range of different outcomes: from full mating compatibility among some populations to complete assortative mating among others. The effectiveness of the methodology in contributing to defining species limits was shown in two species complexes: A. fraterculus and B. dorsalis, and in the case of the latter the synonymization of several established species was published. We conclude that walk-in field cages constitute a powerful tool to measure mating compatibility, which is also useful to determine the role of chemical signals in species recognition. Overall, this experimental approach provides a good source of information about reproductive boundaries to delimit species. However, it needs to be applied as part of an integrative taxonomic approach that simultaneously assesses cytogenetic, molecular, physiological and morphological traits in order to reach more robust species delimitations.
species delimitation, field cages, Tephritidae , Anastrepha fraterculus , Bactrocera dorsalis , Ceratitis fasciventris, Ceratitis anonae , Ceratitis rosa
The biological species concept proposes the occurrence of reproductive isolation barriers that prevent interbreeding and hybridization between species (
The elucidation of the mechanisms that underlie reproductive incompatibility among species within cryptic species complexes is relevant to accurately delimit species and to understand mate recognition systems. According to
One paradigmatic example of the need to resolve species complexes comes from Tephritidae fruit flies. This family is composed of approximately 5,000 species of fruit flies (
Here we aimed to review the value and usefulness of a methodology that assess mating compatibility under standard semi-natural conditions. We focused on the Tephritidae family in general, and on the Anastrepha fraterculus (Wiedemann) cryptic species complex and the Bactrocera dorsalis complex (
The international manual for Product Quality Control for Sterile Mass-Reared and Released Tephritid Fruit Flies (
The standardized Mating Performance Field Cage Test ( |
Mating Performance Field Cage Tests can be applied to assess mating compatibility among two or more populations/species from a given species complex in studies aiming to clarify the taxonomic relationships within species complexes. Data derived from these tests can generate simple, reproducible, meaningful indices of sexual compatibility that can be used to make comparisons between different populations by observing the components of the courtship and mating behavior and any other intra- and inter-sexual interactions during the time of sexual activity. Mating compatibility studies using this standard test gave relevant results for different fruit fly species, including Ceratitis capitata (Wiedemann), species within the FAR complex, species within the B. dorsalis complex, Zeugodacus cucurbitae (Coquillett) and the A. fraterculus cryptic species complex.
For C. capitata,
The Ceratitis FAR complex is composed of three species, C. anonae (Graham), C. rosa Karsch and C. fasciventris (Bezzi) that occur in certain areas of Africa. Due to their highly invasive potential and some difficulties in distinguishing some members of the complex morphologically, a number of different approaches for species recognition were used (reviewed in
A whole body of evidence has been collected that supports the hypothesis that the nominal species Bactrocera dorsalis (Hendel), Bactrocera papayae Drew and Hancock, and Bactrocera philippinensis Drew and Hancock, all belonging to the B. dorsalis complex, are in fact one biological species with geographical variation.
The different host use pattern of Z. cucurbitae observed in Mauritius and some African locations suggested the possibility of a sibling species complex, i.e. species that are the closest relative of each other and have not been distinguished from one another taxonomically (
Anastrepha ludens (Loew) is a species that belongs to the fraterculus group and is a major pest in Mexico and other Central American countries (
Anastrepha fraterculus is a species with a wide geographical range (
Summary of sexual isolation indices from field cage tests carried out for the Anastrepha fraterculus complex.
Reference | Population – mating combination | Morphotypes combination | ISI | Isolation level |
---|---|---|---|---|
Petit-Marty et al. 2004 | Tucumán (Arg) – Entre Ríos (Arg) | Brazilian-1 – Brazilian-1 | –0.01 ± 0.17 | Random mating |
Tucumán (Arg) – Misiones (Arg) | Brazilian-1 – Brazilian-1 | –0.03 ± 0.05 | Random mating | |
Jujuy (Arg) – Tucumán (Arg) | Brazilian-1 – Brazilian-1 | – 0.01 ± 0.05 | Random mating | |
Jujuy (Arg) – Entre Ríos (Arg) | Brazilian-1 – Brazilian-1 | – 0.04 ± 0.17 | Random mating | |
Jujuy (Arg) – Misiones (Arg) | Brazilian-1 – Brazilian-1 | –0.09 ± 0.09 | Random mating | |
Misiones (Arg) – Entre Ríos (Arg) | Brazilian-1 – Brazilian-1 | –0.09 ± 0.13 | Random mating | |
|
La Molina (Peru) – Entre Ríos (Arg) | Peruvian – Brazilian-1 | 0.92 ± 0.03 | High |
Tucumán (Arg) – Piura + La Molina (Peru) | Brazilian-1 – Peruvian | 0.83 ± 0.06 | High | |
Tucumán (Arg) – La Molina (Peru) | Brazilian-1 – Peruvian | 0.82 ± 0.03 | High | |
La Molina (Peru) – Ibague (Col) | Peruvian – Andean | 0.78 ± 0.02 | High | |
La Molina (Peru) – Piracicaba (Br) | Peruvian – Brazilian-1 | 0.55 ± 0.06 | Moderate | |
Tucumán (Arg) – Piracicaba (Br) | Brazilian-1 – Brazilian-1 | 0.43 ± 0.08 | Moderate | |
Tucumán (Arg) – Entre Ríos (Arg) | Brazilian-1 – Brazilian-1 | 0.12 ± 0.10 | Random mating | |
La Molina (Peru) – Piura + La Molina (Peru) | Peruvian – Peruvian | 0.10 ± 0.12 | Random mating | |
|
Tucumán (Arg) – La Molina (Peru) | Brazilian-1 – Peruvian | 0.77 ± 0.05 | High |
Tucumán (Arg) – La Molina (Peru)Unisex Arg | Brazilian-1 – Peruvian | 0.73 ± 0.05 | High | |
Tucumán (Arg) – La Molina (Peru)Unisex Peru | Brazilian-1 – Peruvian | 0.86 ± 0.04 | High | |
Hybrid ArgPeru – ArgUnisexArg | Hybrid Brazilian-1 /Peruvian – Brazilian-1 | 0.30 ± 0.12 | Moderate | |
Hybrid PeruArg – ArgUnisexArg | Hybrid Peruvian /Brazilian-1 – Brazilian-1 | 0.15 ± 0.11 | Random mating | |
Hybrid ArgPeru – PeruUnisexPeru | Hybrid Brazilian-1 /Peruvian – Peruvian | 0.10 ± 0.10 | Random mating | |
Hybrid PeruArg – PeruUnisexPeru | Hybrid Peruvian /Brazilian-1 – Peruvian | 0.13 ± 0.09 | Random mating | |
|
Tucumán (Arg) – Vacaria (Br) | Brazilian-1 – Brazilian-1 | 0.12 ± 0.06 | Random mating |
Tucumán (Arg) – Pelotas (Br) | Brazilian-1 – Brazilian-1 | 0.14 ± 0.09 | Random mating | |
Pelotas (Br) – Vacaria (Br) | Brazilian-1 – Brazilian-1 | 0.14 ± 0.08 | Random mating | |
|
Pelotas (Br) – Bento Gonçalves (Br) | Brazilian-1 – Brazilian-1 | 0. 14 ± 0.07 | Random mating |
São Joaquim (Br) – Vacaria (Br) | Brazilian-1 – Brazilian-1 | 0.04 ± 0.04 | Random mating | |
São Joaquim (Br) – Bento Gonçalves (Br) | Brazilian-1 – Brazilian-1 | 0.14 ± 0.07 | Random mating | |
Bento Gonçalves (Br) – Vacaria (Br) | Brazilian-1 – Brazilian-1 | 0.03 ± 0.05 | Random mating | |
Piracicaba (Br) – São Joaquim (Br) | Brazilian-1 – Brazilian-1 | 0.55 ± 0.09 | Moderate | |
Piracicaba (Br) – Bento Gonçalves (Br) | Brazilian-1 – Brazilian-1 | 0.56 ± 0.05 | Moderate | |
Piracicaba (Br) – Vacaria (Br) | Brazilian-1 – Brazilian-1 | 0.53 ± 0.10 | Moderate | |
|
Xalapa (Mex) – Tucumán (Arg) | Mexican – Brazilian-1 | 0.82 ± 0.06 | High |
Xalapa (Mex) – Vacaria + Pelotas (Br) | Mexican – Brazilian-1 | 0.89 ± 0.02 | High | |
Xalapa (Mex) – La Molina (Peru) | Mexican – Peruvian | 0.74 ± 0.03 | High | |
|
Tucumán (Arg) – Ibague (Col) | Brazilian-1 – Andean | 1 | High |
Xalapa (Mex) – Ibague (Col) | Mexican – Andean | 0.94 | High | |
La Molina (Peru) – Ibague (Col) | Peruvian – Andean | 0.65 | Moderate-High |
There is no doubt that mating compatibility studies carried out in small cages under laboratory conditions can result, because of the high densities and close proximity of flies due to the limited space, in interspecific matings that normally would not occur under natural or even semi-natural conditions. The unreliability of small cage mating tests and the need to develop a field cage method that reflects more the natural situation was realized at the early stages of fruit fly SIT programs (
Since then, the field cage test that is routinely used in SIT programs around the world has gradually evolved (
As a standard index, the Index of Sexual Isolation (ISI, see Box
Although mating compatibility studies in walk-in field cages have been used to delimit species boundaries, there are variants to the standard protocol that can contribute to complete the picture of the potential acting isolation mechanisms. Here we discuss some of these possible variants and provide examples in which these modifications were found to be beneficial.
The general recommendation for the Mating Performance Field Cage Test is to release the flies at a 1:1 male:female ratio. However, this can bias measures of pre-zygotic compatibility for populations which show subtle differences in the timing of mating. For instance, the already mentioned study of
One alternative to avoid the effect of a different timing of sexual activity on mating compatibility is to attempt matching the mating time of flies from different populations. This can potentially be achieved by maintaining the flies during their pre-copulatory period, which depending on the species can vary between 2–30 days, under different light regimes to synchronize the peaks of their mating times. This approach was used by
In many cases, sourcing of the flies can be problematic as often populations, morphotypes, or species can be hard to find in nature or for which collection, transport, export or import permits are difficult to obtain. In those cases, compatibility studies require the establishment of laboratory colonies and this raises concerns on the extent, degree and impact of laboratory adaptation on the sexual behavior of the flies. Reduction of male competitiveness, changes in male courtship, and increase in female receptivity associated to mass-rearing are frequently documented in the literature (
The wide range of mating compatibility studies performed over the last years has generated data that allow assessing the impact of laboratory rearing on mating compatibility. Two populations of B. dorsalis originating from central and southern Thailand showed initially significant positive assortative mating as a result of differences in latency to mate and the degree of male and female fly participation in mating. After one year of rearing under the same laboratory conditions, flies from the same populations mated at random and no differences in latency or female receptivity were detected (
Hybrids derived from populations or morphotypes that show sexual isolation under walk-in field cage conditions, but that can produce progeny under high-density conditions using small laboratory cages, can be a fertile ground to understand speciation processes. For example, male hybrids obtained under such conditions from matings between A. fraterculus populations from Peru and Argentina produced a pheromone that was a mix of the parental pheromones (
The ability to find a mate depends on the recognition between members of the same species. The sensory system is a significant component of sexual communication across many insect taxa and plays an important role in pre-mating isolation (
As in many others, but not all, tephritid flies, A. fraterculus has a lek mating system (
The volatiles emitted by the males seem to vary both in quality and quantity across morphotypes within the A. fraterculus complex (
Chemical profiles of the male-borne volatiles from different populations from the Anastrepha fraterculus complex.
Morphotype | |||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|
Compound | Brazilian-1 | ||||||||||
Tucumán | Tucumán | Bento Gonçalves | Bento Gonçalves | Pelotas | Pelotas | São Joaquim | São Joaquim | Piracicaba | Vacaria | ||
RI | 1 | 2 | 2 | 3 | 2 | 3 | 2 | 3 | 2 | 2 | |
3-Hexanone | 791 | wi | wi | wi | wi | wi | wi | wi | wi | wi | wi |
2-Hexanone | 796 | wi | wi | wi | wi | wi | wi | wi | wi | wi | wi |
Hexanal | 801 | wi | wi | wi | wi | wi | wi | wi | wi | wi | wi |
α-Pinene | 938 | wi | wi | wi | + | wi | ++ | wi | ++ | wi | wi |
Camphene | 956 | wi | wi | wi | wi | wi | wi | wi | wi | wi | wi |
β-Pinene | 985 | wi | wi | wi | wi | wi | wi | wi | wi | wi | wi |
Myrcene | 991 | wi | wi | wi | wi | wi | wi | wi | wi | wi | wi |
Ethyl hexanoate | 996 | wi | wi | wi | wi | wi | wi | wi | wi | wi | wi |
p-Cymene | 1022/1030 | wi | + | + | wi | +++ | wi | + | wi | + | + |
2-Ethylhexan-1-ol | 1029/1030 | wi | ++ | ++ | wi | + | wi | + | wi | + | +++ |
Limonene*2 | 1041/1035 | - | ++ | ++ | +++ | +++ | +++ | +++ | +++ | + | ++ |
5-Ethenyldihydro-5-methyl-2(3H)-furanone | 1044 | wi | wi | wi | wi | wi | wi | wi | wi | wi | wi |
Indane | 1046 | wi | wi | wi | wi | wi | wi | wi | wi | wi | wi |
(Z)-β-Ocimene | 1050/1035 | wi | ++ | + | wi | + | wi | + | wi | ++ | tr |
(E)-β-Ocimene | 1059 | + | wi | wi | wi | wi | wi | wi | wi | wi | wi |
Linalool | 1101 | wi | wi | wi | wi | wi | wi | wi | wi | wi | wi |
(Z)-Nonanal | 1107 | + | tr | ++ | wi | + | wi | + | wi | + | +++ |
Camphor | 1141 | wi | wi | wi | wi | wi | wi | wi | wi | wi | wi |
(Z)-3-Nonen-1-ol*12 | 1159/1158 | +++ | + | ++ | ++ | tr | ++ | + | ++ | + | + |
(E,Z)-3,6-Nonadien-1-ol*12 | 1161/1160 | wi | ++ | +++ | +++ | tr | ++ | ++ | +++ | +++ | + |
Decenal | 1210 | wi | tr | + | wi | + | wi | + | wi | + | + |
Bornyl acetate | 1293 | wi | wi | wi | wi | wi | wi | wi | wi | wi | wi |
(E)-α-Bergamontene | 1435 | - | wi | wi | wi | wi | wi | wi | wi | wi | wi |
(Z)-β-Farnesene | 1448 | wi | wi | wi | wi | wi | wi | wi | wi | wi | wi |
(Z,E)-α-Farnesene*12 | 1495/1492 | +++ | + | + | + | + | — | + | + | + | + |
Germacrene D | 1498/1502 | wi | tr | + | wi | + | wi | + | wi | + | + |
Suspensolide | 1506/1509 | +++ | + | ++ | wi | + | wi | + | wi | ++ | + |
(E,E)-α-Farnesene*12 | 1512/1510 | +++ | +++ | ++ | +++ | ++ | +++ | +++ | +++ | +++ | + |
Anastrephin | 1617/1610 | +++ | + | + | wi | + | wi | + | wi | + | + |
Caryophyllene oxide | 1606 | wi | wi | wi | wi | wi | wi | wi | wi | wi | wi |
Epianastrephin*12 | 1621/1625 | +++ | + | + | + | + | +++ | ++ | + | + | + |
Benzoic acid | wi | +++ | wi | wi | wi | wi | wi | wi | wi | wi | wi |
β-Bisaboline | wi | - | wi | wi | wi | wi | wi | wi | wi | wi | wi |
Morphotype | |||||||||
---|---|---|---|---|---|---|---|---|---|
Compound | Brazilian-3 | Peruvian | Andean | ||||||
Alagoas | Alagoas | Alagoas | La Molina | Ibague | Sibundoy | Duitama | Cachipay | ||
RI | 2 | 4 | 3 | 1 | 3 | 3 | 3 | 3 | |
3-Hexanone | 791 | wi | tr | wi | wi | wi | wi | wi | wi |
2-Hexanone | 796 | wi | + | wi | wi | wi | wi | wi | wi |
Hexanal | 801 | wi | + | wi | wi | wi | wi | wi | wi |
α-Pinene | 938 | wi | + | + | wi | +++ | +++ | +++ | +++ |
Camphene | 956 | wi | tr | wi | wi | wi | wi | wi | wi |
β-Pinene | 985 | wi | + | wi | wi | wi | wi | wi | wi |
Myrcene | 991 | wi | + | wi | wi | wi | wi | wi | wi |
Ethyl hexanoate | 996 | wi | + | wi | wi | wi | wi | wi | wi |
p-Cymene | 1022/1030 | ++ | + | wi | wi | wi | wi | wi | wi |
2-Ethylhexan-1-ol | 1029/1030 | tr | + | wi | wi | wi | wi | wi | wi |
Limonene*2 | 1041/1035 | +++ | +++ | +++ | + | +++ | +++ | +++ | +++ |
5-Ethenyldihydro-5-methyl-2(3H)-furanone | 1044 | wi | + | wi | wi | wi | wi | wi | wi |
Indane | 1046 | wi | + | wi | wi | wi | wi | wi | wi |
(Z)-β-Ocimene | 1050/1035 | — | + | wi | wi | wi | wi | wi | wi |
(E)-β-Ocimene | 1059 | wi | + | wi | +++ | wi | wi | wi | wi |
Linalool | 1101 | wi | tr | wi | wi | wi | wi | wi | wi |
(Z)-Nonanal | 1107 | + | wi | wi | + | wi | wi | wi | wi |
Camphor | 1141 | wi | tr | wi | wi | wi | wi | wi | wi |
(Z)-3-Nonen-1-ol*12 | 1159/1158 | + | tr | ++ | + | + | + | + | + |
(E,Z)-3,6-Nonadien-1-ol*12 | 1161/1160 | +++ | + | +++ | wi | + | + | + | + |
Decenal | 1210 | + | wi | wi | wi | wi | wi | wi | wi |
Bornyl acetate | 1293 | wi | + | wi | wi | wi | wi | wi | wi |
(E)-α-Bergamontene | 1435 | wi | + | wi | +++ | wi | wi | wi | wi |
(Z)-β-Farnesene | 1448 | wi | + | wi | wi | wi | wi | wi | wi |
(Z,E)-α-Farnesene*12 | 1495/1492 | + | + | + | - | + | + | + | + |
Germacrene D | 1498/1502 | + | tr | wi | wi | wi | wi | wi | wi |
Suspensolide | 1506/1509 | ++ | + | wi | +++ | wi | wi | wi | wi |
(E,E)-α-Farnesene*12 | 1512/1510 | + | +++ | — | +++ | + | + | + | + |
Anastrephin | 1617/1610 | + | + | wi | +++ | wi | wi | wi | wi |
Caryophyllene oxide | 1606 | wi | tr | wi | wi | wi | wi | wi | wi |
Epianastrephin*12 | 1621/1625 | + | + | + | +++ | + | + | + | + |
Benzoic acid | wi | wi | wi | wi | - | wi | wi | wi | wi |
β-Bisaboline | wi | wi | wi | wi | +++ | wi | wi | wi | wi |
Walk-in field cages can also be useful to explore the role of chemical communication in mate finding and species recognition and the Manual for Product Quality Control for Sterile Mass-Reared and Released Tephritid Fruit Flies (
To determine whether A. fraterculus male pheromones from two different populations were equally attractive to con-specific and hetero-specific females, the methodology of
For the purpose of evaluating the response of females towards the male pheromone, an indicator of intra-specific recognition in lek-forming tephritids, field cages are set up with two potted trees inside, which are virtually divided into two sectors. Each sector contains one tree. The test involves two steps. In the first, it is determined whether females orient to the pheromone of con-specific males and 25 mature virgin females of a given population are released into the field cage during the period of sexual activity. Fifteen minutes later, 3 “artificial leks” consisting of cylindrical metal wire-mesh containers (3 cm diam., 7 cm long) with 7 sexually mature males inside (Figure |
Results from the Wilcoxon sign rank test to evaluate orientation A. fraterculus females from different populations to artificial leks (containers with sexually mature males).
Area | Tree | Lek | ||||||
---|---|---|---|---|---|---|---|---|
Lek combination | Female | N | Z | p-level | Z | p-level | Z | p-level |
Control |
Piracicaba | 5 | 0,73 | 0,4652 | 2,02 | 0,0431 | 2,02 | 0,0431 |
Control | Tucumán | 8 | 1,12 | 0,2626 | 1,12 | 0,2626 | 2,20 | 0,0277 |
Tucumán – Piracicaba | Piracicaba | 7 | 0,54 | 0,5896 | 1,35 | 0,1763 | 0,40 | 0,6858 |
Tucumán – Piracicaba | Tucumán | 7 | 1,86 | 0,0630 | 0,51 | 0,6121 | 0,53 | 0,5930 |
The results presented above showed that walk-in field cages can be used to measure the response of A. fraterculus females towards volatiles emitted by A. fraterculus calling males and this opens opportunities to better understand the mechanisms behind mating isolation between morphotypes. The experimental protocol, however, entails two issues that need to be resolved: first, the comparisons are restricted to populations that have the same timing of sexual activity; second, it is not possible to control the amount of chemical stimuli released as the number of males that are calling at any particular time within the artificial lek cannot be controlled. The former can be solved by changing the photoperiod of those populations that have different time of sexual activity, while for the latter it is advisable to monitor the number of males calling during the test, as for this species, the numbers of males calling was correlated with the amount of pheromone released (
An alternative approach to solve differences in calling times and in number of calling males is the use of collected volatiles or artificial blends made of synthetics pheromone analogs in the right proportions. Such an approach has been evaluated by
Unlike small laboratory cage tests, walk-in field cage tests have shown to be reliable and powerful tools to measure the level of mating compatibility among different species and populations of a putative single species. This experimental arena under semi-natural conditions can also provide good information on the types of pre-zygotic isolation barriers that contribute to reproductive incompatibility. The intermediate scale of field cages, i.e. between small laboratory cages and open field observations, allows this experimental approach to be implemented in several places and without the need of much infrastructure. It also permits the evaluation of the behavior of flies coming from different regions provided adequate bio-security measures are in place (
Walk-in field cages have also shown their usefulness in determining the role of chemical signals in species recognition. The use of calling males ensures the timely release of the compounds at the right proportions while the use of volatile compounds, instead of male flies, allows evaluating populations in which flies are active at different times of the day. Field cage tests can be accompanied by laboratory tests to assess antennal responses in the females to the various compounds present in the male pheromone. In addition, the role of close distance chemical signals such as cuticular hydrocarbons and other non-chemical signals such as acoustic and visual stimuli remains to be further explored.
Within the context of SIT application, the use of walk-in field cages to asses mating compatibility has not been restricted to the Tephritidae. For tsetse flies see
Overall, the so far attempted evaluation of different testing conditions has shown to provide a better understanding of the pre-zygotic isolation barriers occurring when mating incompatibility is found. As a general recommendation, the most convenient approach in a novel situation is to start with the traditional protocol and only in those cases in which sexual isolation is associated with temporal isolation, adaptations to the standard protocol should be made to evaluate mating compatibility. The experimental approach reviewed here provides a good source of information to delimit reproductive species boundaries. However, in any case it is advisable to follow this methodology as part of an integrative taxonomy approach, including also molecular, physiological and morphological traits in the assessments in order to achieve robust species delimitation.
We are grateful to all FAO/IAEA IPCL staff for the contribution during the research visits at the IPCL, particularly to Thilakasiri Dammalage for assistance with the rearing of the flies and provision of the biological material. We are also very grateful to all Coordinated Research Project (CRP) participants who contributed to stimulating discussions during this five-year’s project. Funding was provided through a Research Contract (16038) as part of the FAO/IAEA CRP on ‘Resolution of cryptic species complexes of tephritid pests to overcome constraints to SIT application and international trade’ as well as a FAO/IAEA consultancy to FD and a mobility project between Argentina and the Czech Republic to MTV (ARC1209) and BK (MSMT CR Mobility 7AMB13AR018). Finally, we wish to add some words in the memory of Dr. Peter Teal. Peter was the first to describe differences at the pheromone level in A. fraterculus and these initial studies triggered a whole world of questions which we are still trying to address. He loved to teach and train students and researchers, and transfer as much as possible his knowledge and his passion for science. We are really grateful to him for all his enthusiasm. We will miss having him around.