2013. Tucuman 3Rd. RCM. August-2013. Produced Working

IAEA-D41023-CR-3 LIMITED DISTRIBUTION

WORKING MATERIAL

RESOLUTION OF CRYPTIC SPECIES COMPLEXES OF TEPHRITID PESTS TO OVERCOME CONSTRAINTS TO SIT APPLICATION AND INTERNATIONAL TRADE

THIRD RESEARCH COORDINATION MEETING OF A FAO/IAEA COORDINATED RESEARCH PROJECT HELD IN TUCUMAN, ARGENTINA FROM 26-31 AUGUST 2013

Reproduced by the IAEA Vienna, Austria 2014

______

NOTE

Material in this document has been supplied by the authors and has not been edited by the IAEA. The views expressed remain the responsibility of the named authors and do not necessarily reflect those of the government of the designating Member State(s). In particular, neither the IAEA nor any other organization or body sponsoring the meeting can be held responsible for any material reproduced in this document. 2

Table of Contents

A. Background Situation Analysis ...... 3 B. The Co-ordinated Research Project (CRP) ...... 4 C. Report for the 3rd RCM (Tucuman 2013) ...... 5 D. Conclusions on Current Status and Recommended Future Activities for the CRP Participants ...... 10 Anastrepha fraterculus Complex ...... 10 Background Situation Analysis ...... 10 Baseline and Progress on Anastrepha fraterculus ...... 12 Specific Progress and Individual Research Plans: Anastrepha fraterculus Complex ...... 19 Bactrocera cucurbitae ...... 30 Background Situation Analysis ...... 30 Baseline and Progress on Bactrocera cucurbitae ...... 30 Specific Progress and Individual Research Plans: Bactrocera cucurbitae .... 33 TAXONOMY...... 33 Bactrocera dorsalis Complex ...... 36 Background Situation Analysis ...... 36 Baseline Knowledge on Five Priority Species in the Bactrocera dorsalis Complex ...... 37 Specific Progress and Individual Research Plans...... 45 Ceratitis FAR Complex ...... 57 Background Situation Analysis ...... 57 Baseline and Progress on the Ceratitis FAR Complex ...... 57 Specific Progress and Individual Research Plans: Ceratitis FAR Complex.. 62 E. Logical Framework ...... 67 F. Workshop on “Reviewing Evidence to Resolve Species Complexes of Tephritid Pests” (31. August 2013) ...... 77 G. Appendices...... 96 Appendix 1: 3 rd RCM Participants ...... 97 Appendix 2: Agenda ...... 108 Appendix 3: Abstracts 3rd RCM Tucuman ...... 113 Appendix 4: Working Groups...... 147 Appendix 5: Protocols for Collection and Shipment of Live and Dead Insects for Vouchering, Rearing, Morphology, Morphometrics, Chemical Ecology and Molecular Assays ...... 148 Appendix 6: Guidelines for Performing Mating Compatibility Field Cage Test ... 151 Appendix 7: Male Sex Pheromonal Components in Bactrocera Species – Extraction of Rectal (Pheromonal) Gland for Chemical Analyses ...... 163 Appendix 8: List of References ...... 169 3

RESOLUTION OF CRYPTIC SPECIES COMPLEXES OF TEPHRITID PESTS TO OVERCOME CONSTRAINTS TO SIT APPLICATION AND INTERNATIONAL TRADE

A. Background Situation Analysis

Tephritids are among the worst pests in agriculture and are of major economic importance in nearly all tropical, subtropical and temperate countries worldwide. These pest species cause enormous devastation to both food production and international trade and are major targets of insecticide applications. They are among primary causes of poverty, malnutrition and poor production and trade in fresh horticultural commodities in large areas of tropical developing countries (Waterhouse 1993; Allwood & Leblanc 1996; Lomborg 2004), where climatic conditions are favourable for labour-intensive fruit and vegetable-based agroindustries.

The study of the biology and management of tephritids therefore requires significant international attention to overcome these transboundary hurdles and to assist Member States in developing and validating environment-friendly suppression systems to support viable fresh fruit and vegetable production and export industries. Such international attention has resulted in the successful development and validation of a Sterile Insect Technique (SIT) package for Mediterranean fruit fly. Demands for R&D support for this pest species are diminishing due to successful integration into control programmes. There is now an urgent need to focus on the increasing demands from Member States in Africa, Asia and Latin America to address other major tephritid species or groups of economic importance. These are groups of species where the morphology is very similar or identical but biologically they are distinct. Some major pest fruit fly species occur within complexes, and the uncertainty related to their questionable technical status results in major constraints to SIT application and international trade.

Problem to be addressed:

Considerable concern has been expressed by many Member States that some major tephritid pest species complexes include taxonomically described species that are actually only geographical variants of the same species. Conversely, some fruit fly populations grouped taxonomically within the same pest species display different biological and genetic traits and show reproductive isolation which suggest that they are different species. This uncertain taxonomic status is having important practical implications on the effective development and use of the SIT against such complexes with respect to the rearing of the correct species and significantly affects international movement of fruit and vegetables, resulting in the establishment of trade barriers to important agricultural commodities which are hosts to pest tephritid species. Moreover, this problematic situation appears to occur in four of the most important pest genera within the Tephritidae family: Anastrepha, Bactrocera, Ceratitis and Rhagoletis .

Regional insect rearing facilities that would produce sterile males for use in different countries or regions are desirable as this would make the SIT more cost-effective. It is of prime importance therefore to the success of these regional area-wide integrated pest management (AW-IPM) programmes that have an SIT component that the mass-reared flies from such species complexes as Anastrepha and Bactrocera, etc. are behaviourally compatible with the target native fruit fly pest populations in the various recipient regions. If 4 species complexes remain unresolved then these desirable outcomes will be difficult or impossible to achieve. The resolution of some of the major cryptic species complexes (i.e. insects whose specific status is questionable) is therefore critical both for SIT application and to assist subtropical and tropical Member States to overcome non-tariff trade barriers in order to export their fresh fruit and vegetable commodities to international high value markets.

A ranking of the major fruit fly pest complexes with respect to the economic damage and regional impacts indicates that a number of them are of concern to Member States. They are:

• Known complexes o Anastrepha fraterculus o Bactrocera dorsalis o Ceratitis anonae, C. fasciventris, C. rosa

• Suspected complex o Bactrocera cucurbitae

A Consultants’ Meeting, held from 6 to 10 July 2009 in Vienna, Austria, discussed the complexes noted above and related technical issues and prioritised them as to economic importance and potential for SIT application. The three complexes and the suspected one were confirmed to be of significant importance that needed to be resolved to facilitate world agricultural trade and SIT programmes.

B. The Co-ordinated Research Project (CRP)

This Co-ordinated Research Project (CRP) is based on the mentioned Consultants Meeting that reviewed the background and scientific progress made in this area, assessed the potential for conducting co-ordinated R&D and formulated a proposal for a CRP on “Resolution of Cryptic Species Complexes of Tephritid Pests to Overcome Constraints to SIT Application and International Trade”.

The overall objective of this new CRP D4.10.23 , approved for the period 2010-2015, is to assist Member States in overcoming constraints to SIT application against fruit flies and in facilitating international trade through the resolution of cryptic species complexes of major tephritid pests.

The specific objectives are: a) to define the species limits within the target complexes, and b) to develop robust species-specific diagnostic tools.

The main targets are the Anastrepha fraterculus (Latin America), Bactrocera dorsalis (Asia & Pacific, Africa), and Ceratitis anonae, C. fasciventris C. rosa (Africa) species complexes and with lower priority a possible Bactrocera cucurbitae (Asia & Pacific, Africa) complex. In each of these groups there are questions concerning either the validity of some of the species or their capacity to be diagnosed.

This international network research project is operated as part of the IAEA Research Contract Programme. The new FAO/IAEA CRP D4.10.23 includes 22 research teams on all continents, of which 15 are research contract holders and 7 are agreement holders. In addition several other research teams are participating fully funded by their institutions and 5 governments. The duration of the CRP is 5 years and Research Co-ordination Meetings (RCMs) are held every 18 months.

These CRPs and RCMs have the objective to encourage networking and close collaboration among institutions in developed and developing countries, and to provide a forum for information exchange between scientists and a focused approach to the development, capacity building and technology transfer of environment-friendly technologies.

The first RCM for this project was held in Vienna from 2 to 6 August 2010 and the second RCM was held at the EcoCentre of Griffith University in Brisbane, Australia from 31 st January to 3 February 2012 (reports available).

C. Report for the 3rd RCM (Tucuman 2013)

Objectives of the Meeting

The objectives of the third RCM were as follows:

a) to present, discuss and review the results of the research carried out by participating researchers since the 2 nd RCM; b) to co-ordinate and harmonize the processing and analysis of the experimental results; c) to discuss the next activities and agree on protocols for the final phase of the CRP; d) to prepare work plans for each participating institute for the final phase of the CRP; e) to co-ordinate the training and other needs and provision of material over the next 18 months; and f) to strengthen research and administrative linkages between research groups and institutions.

The meeting was attended by 52 scientists and observers from 18 countries. The list of 3rd RCM participants is given in Appendix 1. The meeting was formally opened by the host, Teresa Vera of Universidad Nacional de Tucumán, who welcomed the participants and described the logistics and organisational aspects. Jorge Hendrichs, as Scientific Secretary of the CRP, explaines the objectives of the meeting, the importance of resolving the issues that surround the existence of cryptic Tephritid species complexes and reviewed the agenda of the 3rd RCM and of the workshop on the final day with an external panel of experts on “Reviewing Evidence to Resolve Species Complexes of Tephritid Pests” (see Section F ).

The agenda that was prepared for the RCM (Appendix 2) was followed with only minor changes. The progress reports of the participants covered the results they obtained during the 18 month since the 2 nd RCM of the CRP. The abstracts of these presentations are given in Appendix 3.

Between the 3 rd and 5th days of the RCM participants were divided into three separate working groups ( Appendix 4) to review overall achievements based on original coordinated research plans, to assess results and scientific/technical conclusions for the different genera, thematic areas/approaches and techniques, and to review research needs to be addressed and other activities to be implemented. Also the progress made by the individual research teams was evaluated and research proposals/plans for the final 18 months discussed for the different genera and approaches/thematic areas. In addition, cross-cutting research and the scientific and administrative linkages between participating institutes were detailed. 6

Protocols for collection and shipment of live and dead insects for rearing, morphology, chemical biology and molecular assays and procedures for field cage tests (Appendices 5, 6, and 7) were updated. Relevant references are presented in Appendix 8.

Working Groups

The three working groups that were established based on pest fruit fly groups being worked on were:

Group 1: Anastrepha fraterculus (possibly five to seven distinct pest species)

Group 2: Bactrocera dorsalis pest species complex ( B. dorsalis s.s. , B. carambolae , B. papayae , B. philippinensis and B. invadens , some of which are not distinct species)

Group 3: Bactrocera cucurbitae (possibly different populations) and the Ceratitis FAR pest species complex (comprising at least three species: C. fasciventris , C. anonae and C. rosa ).

Group participants firstly reviewed the progress made since the 2 nd RCM in addressing the identified gaps in each thematic area in relation to the baseline knowledge determined at the start of the CRP. In addition to this, participants in each group also provided each research group’s specific progress made since the 2 nd RCM and the specific work plans for the final 18 months of the CRP.

Conclusions of the three Working Groups are reported in Section D. A summary of thematic areas in relation to the species complexes being addressed by researchers is presented in Table 1 . Given that a number of researchers and institutions are involved in various sub- projects within the CRP, Table 2 was updated to facilitate identifying these project linkages and interactions.

Working Groups then reviewed their inputs into the Logistic Framework ( Table 3 ) to ensure that targets previously set were reasonable and feasible both technically and with respect to the duration of the CRP. This is reported in Section E.

After these tasks were completed all Working Groups rejoined to present and discuss any changes and new inputs and agreed on the content on a draft Working Material for the 3rd RCM of this CRP.

During the final day (August 31 st 2013) a workshop was held with an external panel of independent experts to review all the evidence collected so far during the CRP to resolve species complexes of tephritid pests. The resulting summaries and character matrices for each of the species complexes are presented in Section F .

Table 1. Thematic areas in relation to Tephritid species complexes and suspected complexes being addressed by researchers

Thematic area

Complex Molecular Behaviour Chemical Taxonomy / Ecology Morphology

Anastrepha Canal Abraham Brizova Bartolucci fraterculus Castañeda Dias Mendonça Canal Cladera Joachim-Bravo Nascimento Hernández-Ortíz De Lima Kovaleski Vanickova Selivon Drosopoulou Mendonça Segura Steck Malacrida Nascimento Teal Passos Roriz Paranhos Selivon Rull Silva Vera Steck Vilardi Zacharopoulou Bactrocera Aketarawong Caceres Chinvinijkul Chen dorsalis Armstrong Amornsak Hee Clarke Augustinos Chinvinijkul Kalinova Drew Barr Clarke Tan Jessup Drosopoulou Ekesi Teal Ji Hasanuzzaman Haq Wee Leblanc Haymer Hendrichs Romig Khamis Hee Steck Malacrida Ji Schutze Thanapum Schutze Tsuruta Yesmin Shelly Zacharopoulou Srikacher Tan

Ceratitis Delatte de Meyer Kalinova de Meyer FAR Haymer Ekesi Steck complex Khamis Mwatawala Quilici Quilici Virgilio Bactrocera Delatte Delatte de Meyer cucurbitae Malacrida Ekesi - Leblanc Virgilio Haq Zacharopoulou Mwatawala Quilici

Table 2. Project linkages

Thematic area Behaviour Taxonomy, (pre,post Host Participant Country DNA Cytology Genomics Morphology/metrics Zygotic) Pheromones/Hydrocarbons range Distribution

Aketarawong/Sujinda Thailand New Armstrong Zealand

Bravo/Diaz/Beatriz Brazil Canal/Castañeda Colombia Clarke/Schutze Australia

de Meyer/Virgilio Belgium

Delatte/Quilici Reunion

do Nascimento / Dias/Adriana/Beatriz Brazil Drew/Romig Australia

Ekesi / Khamis / Mohamed Kenya

Hee/Wee/Nishida/Tan Malaysia

Malacrida, Gasperi Italy Mwatawala Tanzania Mwatawala Tanzania Hernandez Mexico Qinge China Silva Brazil

Steck USA

Suksom/Sunynee Thailand

Tan/Hee/Wee Malaysia 9

Vanickova/Kalinova Czech

Vanickova/Kalinova Czech Vera Argentina

Zacharopolou Greece Barr USA Barr USA

Seibersdorf FAO/IAEA

Bactrocera cucurbitae

Ceratitis FAR complex

Anastrepha fraterculus

Bactrocera dorsalis complex

D. Conclusions on Current Status and Recommended Future Activities for the CRP Participants

Anastrepha fraterculus Complex

Background Situation Analysis The South American fruit fly, Anastrepha fraterculus (Wiedemann) s.l . is present in most countries of the Americas from the USA to Argentina (Hernández & Aluja 1993, Steck 1999, Zucchi 2000, 2007). On the South American subcontinent, this species is found in two broad, possibly unconnected bands: one along the western edge, including both highland and lowland areas of the Andean range, and the other along the east coast. However, there are recent data of its presence in parts of the Brazilian Amazon basin (Zucchi et al. 2011). Its centre of diversity is South America and it has been reported to infest about 80 host plants including major fruit crops (Norrbom & Kim 1988, Norrbom 2004, Zucchi 2008). This highly destructive pest imposes quarantine restrictions for fruit export to many countries (Steck 1999).

The high levels of variability found among different populations throughout the geographical range of A. fraterculus have led to the conclusion that it is a complex of cryptic species rather than a single biological entity (Stone 1942, Morgante et al . 1980, Solferini & Morgante 1987, Malavasi & Morgante 1982, Steck 1991, Steck & Sheppard 1993, Selivon 1996, Hernández- Ortíz et al. 2004, 2012). Differences have been reported based mainly on morphology, pest status and genetics (including karyotype, isozyme and molecular analyses); these are reviewed in Steck (1999) and some aspects discussed in subsequent studies (McPheron et al . 1999, Norrbom et al . 1999, Silva 2000, Smith-Caldas et al . 2001, Hernández-Ortíz et al . 2004, Selivon et al . 2005, Goday et al . 2006, Prezotto 2008, Cáceres et al . 2009, Barr et al . 2005, Silva & Barr 2008, Selivon & Perondini 2007, Ludeña et al. 2010). However, in order to establish the existence of different species, it is necessary to systematically correlate these genetic and morphological differences with the existence of reproductive isolation and other life history related traits (hosts, demography, etc).

Reproductive incompatibility has been reported both at pre- and post-zygotic levels (Selivon 1996, Vera et al . 2006, Cáceres et al . 2009) among some populations. At the pre-zygotic level, mating compatibility was evaluated among six different populations from Mexico, South America, involving lowland (Peru) and highland (Colombia) areas from the Andean region, and the south-eastern part of the continent (Brazil and Argentina). Most of the populations were shown to have some level of incompatibility with each other and thus sexually isolated. Flies were sexually active at different times of the day suggesting different sexual behaviour. Hybrid females tended to mate with hybrid males and as a result Segura et al. (2011) suggested that hybridization is a possible speciation mechanism.

Post-zygotic studies between two populations from Brazil (Selivon et al . 1999) and between one Argentine population and one Peruvian population (Cáceres et al . 2009) found partial hybrid inviability and sex ratio distortion confirming the existence of post-zygotic barriers. In the former case, cytological, isozyme and molecular studies revealed differences among groups (Malavasi & Morgante 1982, Selivon 1996, Selivon et al . 2005, Goday et al . 2006); while for the latter case, differences between groups were also found in terms of male sex pheromones and karyotypes (Cáceres et al . 2009). 11

The combined results of these studies (see incomplete table below to be further developed and refined with increasing evidence) suggest the existence of at least seven different biological entities which in this document are referred to as: A. sp. 1 aff. fraterculus (Yamada & Selivon 2001), A. sp. 2 aff. fraterculus (Yamada & Selivon 2001), A. sp. 3 aff. fraterculus (Selivon et al . 2004), A. sp. 4 aff. fraterculus (Selivon et al . 2004), A. fraterculus Mexican morphotype (Hernández-Ortíz et al . 2004) and A. fraterculus from Andean highlands and Venezuela coastal lowlands (Steck 1991). A recent comprehensive morphological study supports the existence of these seven morphotypes (Hernández-Ortíz et al. 2012).

Morphotype Population/s Reference Comments (according to Hernández-Ortiz) Brazilian 1 Tucuman, Posadas, Yamada & Selivon A. sp. 1 aff. Concordia (all from 2001 fraterculus sensu Argentina), Vacaria, Hernández-Ortiz Selivon Sao Joaquim, 2012 Pelotas, Piracicaba (all from Brazil) Brazilian 2 Brazil Yamada & Selivon A. sp. 2 aff. 2001 fraterculus sensu Selivon Sympatric morphotypes? Brazilian 3 Brazil Selivon et al . 2004 A. sp. 3 aff. fraterculus sensu Selivon Sympatric morphotypes? Peruvian Peru, Ecuador Selivon et al . 2004 A. sp. 4 aff. fraterculus sensu Selivon Sympatric morphotypes? Andean Colombia highlands Steck 1991 Sympatric Hernández-Ortíz et morphotypes? al. 2012 Venezuelan Venezuela lowlands Steck 1991 Hernández-Ortíz et al. 2012 Mexican Mexico and Central Hernández-Ortíz et America al . 2004 Hernández-Ortiz et al. 2012

Despite previous studies, which have provided strong evidence supporting the existence of several species, there are still major knowledge gaps (see below). Moreover, the described studies have used different methodologies, have not used the same identified biological material, and most importantly, they did not include all of the morphotypes. In order to be able to formally describe and name these putative species, it is necessary to apply a complete 12 set of methodologies to all of them in a comprehensive study involving populations from the entire geographic distribution range. This will allow the characterization of each putative species and will provide sound diagnostic tools for addressing the related management and trade issues.

Definition of species limits and formal naming of these putative species will be critical for importing authorities in determining which of them may or may not be quarantine pests. This will immediately allow some countries to gain access to international fresh fruit markets for those countries and commodities which can be determined to be outside the geographic and host range of correctly delimited A fraterculus s.s. Implementation of the SIT also requires a thorough understanding of species boundaries and sexual compatibility.

Baseline and Progress on Anastrepha fraterculus

DNA - Knowledge at the start of the CRP: • Only three gene regions have been studied for representatives of A. fraterculus s.l. populations. These are the COI, 16S, and period genes. COI and period genes are most useful in terms of inter-population variation. Data is available for only 24 specimens from only 16 populations representing five of the above mentioned seven morphotypes (see Table below):

Number of specimens examined for each morphotype and gene region

Morphotype / biological COI 16S Period entities from:

SC - 2 - SP - 1 Brazil, Argentina, other ES - 1 countries?* BA -1 SP - 2 MG -1 ARG - 1 Peruvian (= A. sp. 4 aff - - - fraterculus of Selivon) Mexican 3 - 2

Andean 3 1 4

Venezuelan lowland 1 - 1

* For some populations there are still no data that confirm the morphotype. Therefore it is advisable to confirm all those populations that are used for behavior, pheromones or other studies.

- Gaps identified at the start of the CRP: • There are no data yet for several of the morphotypes. • The total number of specimens analyzed so far is small. • Microsatellite data are not yet available. • Other molecular markers have not been applied.

- Progress up to the 3rd RCM in addressing identified gaps: • Extensive sampling was performed in Colombia (9 populations), Peru (2 population), Bolivia (1 population), Panama (1 population) and Brazil (34 populations) which resulted in the collection and preservation of more than 350 individuals. • Samples from the colonies established at Seibersdorf were also preserved for DNA extraction. • DNA was extracted and voucher specimens preserved. CO I data was obtained from Brazil (34 populations), Mexico (one population) and Argentina (one population); CO I, CO II and ITS I were screened in Colombia (9 populations); ITS I was screened in Bolivia (1 population), Peru (2 populations), Venezuela (2 populations) and Brazil (1 population). • Mitochondrial markers (CO I, CO II, ITS-I) were used to study 9 Colombian populations (5 individuals/pop, 45 total) and compared with individuals from Ecuador, Brazil, Venezuela, Mexico. Eight of the Colombian populations appeared highly related to each other, but individuals from Putumayo (south near to Ecuador) appeared to be the most differentiated and related with an Ecuadorian population. • Sequencing of Y chromosome is under progress. • Populations from Ecuador were also analysed (Ludeña et al. 2010). • Eighteen microsatellite loci sequences were isolated. • 15 SSR easy to score were validated and characterized for their PIC (polymorphic information content) in two strains (Vienna, Tucuman) and two populations from Argentina (Concordia, Puerto Yerúa). • Some of these microsatellites are being used for the analysis of six Brazilian populations (from Northeast to South). • 90 ISSR loci were developed. Fourteen of them were used to detect host differentiation between synchronic and sympatric samples from peach, guava and walnut in Tucuman region. Strong differences were observed between flies collected from peach and walnut. • Mitochondrial CO I markers have been used to assess the phylogenetic relationships between 244 individuals collected from 34 Brazilian populations (collected across the country from Northeast to South) and from Mexico (Xalapa) and Argentina (Tucuman). A total of 43 haplotypes have been identified which can be clustered in seven haplogroups. The geographic distribution of haplogroups is not homogeneous.

CYTOLOGY - Knowledge at the start of the CRP: • Karyotypes have been described for at least 24 species of Anastrepha . • These include the following morphotypes of A. fraterculus s.l. : Mexican, A. sp. 1 aff fraterculus (Argentina, Brazil), A. sp. 2 aff fraterculus (Brazil), A. sp. 3 aff fraterculus (Brazil), and A. sp. 4 aff fraterculus (Peruvian morphotype from Peru and Ecuador). • Polytene chromosomes were used to compare Argentine and Peruvian populations. - Gaps identified: • Polytene chromosome banding patterns not yet described. • No karyotype data available for Andean and Venezuela lowland morphotypes. - Progress up to the 3rd RCM in addressing identified gaps: • Polytene chromosome banding patterns was described for A. sp. 1 aff fraterculus from Argentina (Giardini et al. 2009). • Karyotypes from 8 populations from Colombia (Andean morphotype) were described.

ALLOZYMES - Knowledge at the start of the CRP: • Extensive data on allozyme variation are available for various populations of the following morphotypes: Mexican, Andean, Peruvian, Venezuelan lowland, A. sp. 1 aff fraterculus (Argentina, Brazil) and A. sp. 2 aff fraterculus (Brazil). - Gaps identified: • As data are not directly comparable between the various studies performed in different laboratories, it is not a priority to gather new isozyme data. - Progress up to the 3rd RCM in addressing identified gaps: • No work conducted as not priority.

MORPHOLOGY - Knowledge at the start of the CRP: • Published morphometric data indicate that at least three morphotypes can be recognized in continental America (“Mexican”, “Andean” and “ A. sp. 1 aff fraterculus ”), based on discriminant function analysis of particular characters from the aculeus, wing and mesonotum (Hernández-Ortíz et al . 2004). • Unpublished data (Hernández-Ortíz et al . in prep.) suggest the presence of four additional morphotypes called “Venezuelan”, “Peruvian” 4 aff fraterculus (Peru and Ecuador), “A. sp. 2 aff fraterculus ” (Brazil) and “ A. sp. 3 aff fraterculus ”(Brazil). • Additional morphometric data using aculeus and wing characters show differences between the types A. sp. 1 aff fraterculus (Brazil) and A. sp. 2 aff fraterculus (Brazil). • Egg morphological differences have been described between the three Brazilian morphotyes (Selivon & Perondini 1998, Selivon et al. 2004). • Only preliminary descriptions of larval stages are available (Steck et al. 1990, Frias et al . 2006). - Gaps identified: • Formal taxonomic descriptions of Mexican, Andean, and “ A. sp. 1 aff fraterculus ” (Brazil) morphotypes are not yet published. • None of the larval stages has been thoroughly described and compared among morphotypes. • Egg morphology is unknown for four morphotypes (Andean, Venezuelan, Mexican and Peruvian). - Progress up to the 3rd RCM in addressing identified gaps: • A study including the seven adult morphotypes using a multivariate approach was published (Hernandez-Ortiz et al ., 2012). • A morphometrics analysis involving 18 populations from Ecuador and Colombia were carried out. An 8 th morphotype was recognized in Ecuador. Eggs were sent to Denise Selivon for description. • SEM observations on third instar larvae have been made on five populations representing three morphotypes (Mexican, Peruvian and A. sp. 1 aff fraterculus ). • A morphometric study of the mouthparts of third instar larvae from the Andean morphotype was carried out. • Egg morphology of several Anastrepha species from the fraterculus group was described (Dutra et al. 2011a). • Second and third instar larval morphology from some species (Anastrepha bahiensis, Anastrepha coronilli and Anastrepha turpiniae ) from the fraterculus group were described (Dutra et al., 2012). 15

• The morphometric analysis of several Brazilian populations was initiated to identify the corresponding morphotype. • New collections of A. fraterculus larvae and adults from Peru and Panama were made. SEM studies on other members of fraterculus group were performed ( A. suspensa and A. obliqua ).

PHEROMONE AND CUTICULAR COMPONENTS / CHEMICAL ECOLOGY - Knowledge at the start of the CRP: • Pheromone composition has been described so far for only two populations ( A. sp. 1 aff fraterculus , as well as probably A. sp. 4 aff fraterculus from Peru) with different methodologies. • Chemicals released by calling males were first investigated by Lima et al . (2001) using a single population from Pelotas, Rio Grande do Sul, Brazil (morphotype to be defined). These chemicals were identified as alcohols, mono- and sesquiterpenes and three isomeric lactones. Later, Caceres et al. (2009) showed that a population from Argentina differed in its composition from a population from Peru (probably A. sp. 4 aff fraterculus ). Preliminary studies carried out on the response elicited by the pheromone produced by A. fraterculus males from an Argentine laboratory population have shown that female antennae respond to six out of thirteen compounds identified as volatiles released by calling males of this population, and these chemicals were identified as: (Z)-3-nonenol, (E,Z)-3,6-nonadienol, geranyl acetone, alfa-farnesene and (S,S)-epianastrephin. • There are few studies concerning hydrocarbon composition in pupae and adults of A. fraterculus . • Preliminary studies performed by Vanickova et al. (2012) have shown that there are quantitative and qualitative differences in the cuticular hydrocarbon composition between virgin males and females depending on age (Argentine laboratory population). • Qualitative and quantitative differences were observed in the chemical composition of cuticular hydrocarbons according to the age of the fly. - Gaps identified: • The composition of the pheromone is not known for all morphotypes. • The function of the identified chemicals either alone or combined in eliciting attraction of conspecific females towards males is still unknown. • Antennal response has been studied in only one Argentine population. • Cuticular hydrocarbon composition is known only from virgin male and females from one Argentine population and one southern Brazil population. - Progress up to the 3rd RCM in addressing identified gaps: • The pheromone volatiles from six Brazilian laboratory F2 populations (Bento Gonçalves, Alagoas, Piracicaba, Vacaria, Pelotas, Sao Joaquim), one Argentinean population (Tucuman, Brazilian 1 morphotype), one Peruvian population (Piura + La Molina Peruvian morphotype), and from hybrids of a cross between the Peruvian and Argentinean population were characterized. • The chemical characterization of the pheromone volatiles from one population from Mexico (Mexican morphotype) and one population from Colombia (Tolima, Andean morphotype) was initiated and is in progress. • Cuticular hydrocarbons from males and females from one Argentinean (Tucuman, Brazilian 1 morphotype), males from four Brazilian population (Bento Gonçalves, Alagoas, Piracicaba and Vacaria), males and females from one Peruvian population 16

(Piura + La Molina, Peruvian morphotype), males from one population from Colombia (Tolima, Andean morphotype), and males and females from one population from Mexico (Mexican morphotype) were characterized.

BEHAVIOUR - Knowledge at the start of the CRP: • A. fraterculus s.l. has a lek mating system. • Mating occurs primarily in the early morning, with some populations showing different mating times (midday for Peruvian population and late afternoon for Colombian population (respectively A. sp. 4 aff fraterculus and Andean/Venezuela Highland morphotypes sensu Vicente 2012). • Pre-zygotic isolation was detected among populations at the continental level. • Male courtship has been described for one Argentine population. • There is some evidence suggesting that populations confined together in field cage trials form segregated leks. • For one Argentine population, it has been shown that female post-copulatory behaviour depends on attributes of the first male suggesting that female decision on remating depends on the conditions of the first male. - Gaps identified: • Mating compatibility has not been evaluated among all morphotypes. • Mating behaviour has been described for only 3 morphotypes. • Factors involved in pre-zygotic isolation are unknown (temporal, ecological or sexual isolation, role of pheromones in mate recognition/acceptance). • Male courtship, male-male interactions and female responses within and among each putative species is not completely known. • The factors that determine lek location should be determined (environmental vs. avoidance of mixed leks). • Female post-copulatory behaviour after mating with males from other populations is unknown. - Progress up to the 3rd RCM in addressing identified gaps: • Mating compatibility between the Mexican morphotype and the Peruvian morphotype as well as between the Mexican morphotype and two populations from Brazil (Vacaria and Pelotas, presumably Brazilian 1 morphotype) and one population from Argentina (Brazilian 1 morphotype) was evaluated. • Mating compatibility evaluations among four populations from southern Brazil (Bento Gonçalves, Pelotas, Vacaria and São Joaquim) showed that southern populations are compatible. A detailed behavioural analysis including calling and mounting sound analysis showed similarity between these populations supporting the mating compatibility tests. • Mating compatibility tests between a southeast population from Brazil (Piracicaba) and three southern populations (Vacaria; São Joaquim and Bento Gonçalves) showed partial incompatibility. Courtship behaviour analysis showed some differences in the calling and fanning behaviour of Piracicaba compared with some southern strains. Sounds analysis showed a higher fundamental frequency in calling (long pulses) and mounting signals of Piracicaba compared to the southern populations. • Mating compatibility tests between Parnamirim (a Brazilian northeastern population, morphotype to be confirmed) and Bento Gonçalves (a Brazilian southern population, morphotype to be confirmed) showed compatibility between them. 17

• Preliminary results of the sequence of the different components from the courtship behavior suggested a different pattern in Parnamirim strain (northeast Brazil) compared with other strains from the south (Bento Gonçalves) and the southeast (Piracicaba). • High levels of sexual isolation were detected between the Mexican morphotype and the populations from southern Brazil and Argentina, even though they are sexually active at the same time (i.e. not temporally isolated). • Mating compatibility tests were performed between males and females of the Andean morphotype (Colombia, Ibague) and males and females of the Brazil 1 morphotype (Argentina), the Mexican morphotype (Mexico, Xalapa) or the Peruvian morphotype (Peru, La Molina). Marked temporal differences in calling activity were detected: the Andean morphotype mated at dusk, the Mexican and Brazil 1 morphotype early in the morning, and the Peruvian morphotype around midday. • Mating compatibility tests between two recently established laboratory strains from Colombia revealed sexual compatibility. • Remating propensity and duration of the refractory period of Argentine and Peruvian females were different yet independent of male origin; however there was a tendency for Peruvian females mated with Argentine males not to allow sperm transfer.

DISTRIBUTION - Knowledge at the start of the CRP: • A. fraterculus s.l. is widely distributed from southern Texas to northern Argentina in South America, but the detailed regional patterns for the 7 morphotypes are not well understood. • Current unpublished information reveals that the Mexican morphotype extends from Mexico to Central America; the Venezuelan morphotype occurs only in one area of the Caribbean lowlands of Venezuela; the Andean morphotype is found in the Venezuelan and Colombian highlands; while the Peruvian morphotype ( A. sp. 4 aff fraterculus ) has been found in at least three localities from Ecuador and Peru. • The distribution of the three Brazilian types is not well known and some of them occur in sympatry. - Gaps identified: • The detailed distributions of all morphotypes in South America are unknown. • Elevational transects in Andean countries are lacking and needed to determine limits of highland and lowland morphotypes. - Progress up to the 3rd RCM in addressing identified gaps: • Information was published on the distribution of A. fraterculus morphotypes: Mexican morphotype extends from Mexico to Central America; the Venezuelan morphotype occurs only in one area of the Caribbean lowlands of Venezuela; the Andean morphotype is present in the Venezuelan and Colombian highlands; while the Peruvian morphotype is found in at least three localities from coastal areas of Ecuador and Peru (Hernandez-Ortiz et al., 2012). • Distribution of the Andean morphotype in Colombia was published (Castañeda et al. 2010), with no records below 900 m elevation. • The extensive sampling performed for DNA studies in Brazil will allow updating the distribution of the morphotypes found in this country. • Morphometric studies revealed the presence of two morphotypes in Ecuador: the Peruvian morphotype in the low lands and a new 8th morphotype (Ecuadorian) in the Andean Valleys. 18

• Surveys in Peru showed that the fraterculus cryptic species complex occurs in mid-to- high elevations of eastern Andean valleys of central Peru. ITS I sequence data for these Peruvian Andean populations and a population from Santa Cruz, Bolivia, match that of the Brazilian 1 morphotype.

HOST RANGE - Knowledge at the start of the CRP: • There are numerous host records available for A. fraterculus s.l. , but these have not been associated with the various morphotypes. • Host lists have been published for several countries, e.g.,Venezuela (Hernández & Morales, 2004), Brazil (Zucchi 2007, Zucchi 2008). - Gaps identified: • Host ranges for most morphotypes are unknown. • Update and revision of available host lists to correct errors is needed. - Progress up to the 3rd RCM in addressing identified gaps: • In those areas where only one morphotype is present (Mexico and Central America, highland Venezuela and Colombia, Argentina), the host range is being updated based on existing records. • Data on two years survey from different hosts was published for Colombia (Castañeda et al. 2010) and afterwards expanded with data from entomological collections. • New host plant information has been obtained in Ecuador and Peru.

POST-ZYGOTIC COMPATIBILITY - Knowledge at the start of the CRP: • Crosses between A. sp . 1 aff fraterculus and A. sp 2 aff fraterculus resulted in a reduction of egg hatch and larval viability and also a distorted sex ratio. • The same has been observed in crosses between populations from Peru and Argentina. • It is possible to maintain hybrid lines, including those with their initial sex ratio distortion maintained, under artificial (laboratory) rearing conditions. • The occurrence of Wolbachia was confirmed for three populations (Brazil, Argentina, Peru (morphotypes to be defined). - Gaps identified: • The occurrence of hybrid sterility has not been evaluated. • The role of Wolbachia in post-zygotic incompatibility has not been determined. - Progress up to the 3rd RCM in addressing identified gaps: • Compatibility among one population from Argentina (Tucuman, Brazilian 1 morphotype) and two populations from Brazil (Vacaria and Pelotas, morphotypes to be defined) was demostrated. • Females of the Mexican morphotype caged with Peruvian or Brazil 1 (Tucuman) morphotype males either did not lay eggs or laid unviable eggs. Brazil 1 morphotype females mated to Mexican morphotype males laid eggs that hatched in significantly lower proportion than those of females mated to homotypic males. The other heterotypic crosses produced eggs equally viable to those laid by females mated with homotypic males. • Colombian morphotype females mated with Mexican, Peruvian or (putative) Brazilian 2 morphotype males laid eggs with significantly lower hatch rate than those laid by females mated with homotypic males. Heterotypic crosses involving Colombian morphotype males produced numerically lower egg hatch than homotypic crosses but 19

the difference was not significant. The cross of F1 x F1 males and females resulted in high fertility levels. • Preliminary results of postzygotic tests with Brazilian populations have suggested no postzygotic isolation among strains from different regions (Piracicaba vs. Bento Gonçalves, Bento Gonçalves vs.Parnamirim and Parnamirim vs. Piracicaba) . • Postzygotic tests with six Colombian populations showed no isolation among them.

Specific Progress and Individual Research Plans: Anastrepha fraterculus Complex

DNA Anna Malacrida - 5 year plan: • Identify SSR markers in different populations possibly representing specific entities across the A. fraterculus complex range and evaluating their degree of differentiation. • Develop a database for population variability / differentiation within the A. fraterculus complex. • Assess the presence of post-zygotic isolation within A. fraterculus entities. • Develop species-specific molecular markers as diagnostic tools for A. fraterculus entities. - Plans for the first 18 months: • Develop SSR markers in A. fraterculus for one morphtype. • Assess if it’s transferable to other morphotypes. - Plans for the second 18 months: • Validate the microsatellite loci sequences isolated. • Assess intra-morphotype variability. • Assess if they are transferable to other morphotypes in collaboration with Janisete Silva. - Plans for the third 18 months: • Define and classify the microsatellites alleles for the 15 loci. • Perform population genetic analysis of Argentinean and Brazilian populations using 15 highly informative loci in cooperation with INTA (Argentina) and UESC (Brazil). • Characterize the Y chromosome from a strain of A. fraterculus from Argentina (Brazilian 1 morphotype). • Assist people to develop ITS 2 markers. - Progress made up to 3 rd RCM: • Eighteen microsatellite loci sequences from A. sp . 1 aff fraterculus were isolated. • 15 SSR have been validated and characterized for their PIC (polymorphic information content) in two strains (Vienna, Tucuman) and two populations from Argentina (Concordia, Puerto Yerua). • Intrapopulation variability and differentiation between the two populations and the two strains have been assessed.

Janisete Silva - 5 year plan: • Obtain samples from A. fraterculus populations in Brazil, including colonies already established. • Evaluate DNA variation using mitochondrial and nuclear gene sequences. • Compare populations for evidence of population structure and potentially distinct gene pools. 20

• Test the monophyly of the A. fraterculus complex using multiple specimens from each putative cryptic species. • Elucidate the number of putative species in the A. fraterculus complex in Brazil. • Map haplotype distribution in Brazil to guide future control programs. • Perform a morphometric analysis to some of the Brazilian populations in collaboration with Vicente Hernández-Ortíz. • Describe immature stages in collaboration with Gary Steck. - Plans for the first 18 months: • Obtain collections of different populations of A. fraterculus in Brazil as well as some other related Anastrepha species for the phylogenetic analysis. • Screen populations and already established colonies for one mitochondrial DNA marker (COI). - Plans for the second 18 months: • Continue collections of A. fraterculus populations. • Incorporate additional DNA markers. - Plans for the third 18 months: • Assign a morphotype to each of the populations under study, by means of morphometric analysis (Top priority). • Complete the ITS-1 analysis (and ITS-2 if possible), including as many morphotypes as possible (Top priority). • Validate the usefulness of the 15 microsatellites already developed for morphotype 1 to the other morphotypes using the same approach as in the FAR complex (in collaboration with Pavia). This point is given top priority. • Perform a population genetic analysis with wild populations from as many morphotypes as possible from Brazil and other countries if available. (Top priority) • Submit an article for publication with the complete study of populations from south and south east Brazil (in collaboration with Iara Joachim-Bravo). • Perform morphometric analysis for at least 10 Brazilian populations in collaboration with Iara Bravo and Vicente Hernandez-Ortiz. • Sequence Wolbacchia to determine the lineages present in samples from different locations in Brazil - Progress made up to 3 rd RCM: • Collections of 33 populations of A. fraterculus in Brazil as well as some other related Anastrepha species were done, totaling 74 species and 630 sequences were obtained for the phylogenetic analysis (COI). • A total of 27 populations from 8 countries were also sampled and screened. • Sequences of COI for 244 A. fraterculus females of 34 populations from Brazil, one population from Argentina (Tucuman) and one population from Mexico (Xalapa). • Two mitochondrial regions (COI and ND6) and six nuclear regions (28S, ITS2, CAD, Period, TGO and TPI) were tested for 111 Anastrepha samples in collaboration with University of Texas-Pan American. • Five Brazilian populations of A. fraterculus were screened for 10 microsatellite loci in collaboration with Silvia Lanzavecchia, INTA (Argentina) and Anna Malacrida, Pavia (Italy).

Nelson Canal - 5 year plan: • Analyze ITS-1 from different populations of A. fraterculus and A. obliqua from Colombia. 21

- Plans for the first 18 months: • Obtain samples and optimize ITS amplification. - Plans for the second 18 months: • Continue collections of A. fraterculus populations. • Perform ITS-1 analyses. - Plans for the third 18 months: • Finish the study of A. fraterculus and A. obliqua with mitochondrial markers CO I, CO II and ITS-1, for Colombian populations (in collaboration with Denise Selivon USP, Brazil). - Progress made up to 3 rd RCM: • Samples were obtained from 5 populations of A. fraterculus and 4 of A. obliqua from Colombia. • ITS-1 amplification technique was optimized. • Nine populations of A. fraterculus across the country were collected. • ITS-1 analysis for eight populations is in progress (sequences are in analysis). • 11 populations of A. obliqua were collected and analysis of CO I, CO II and ITS-1 is in advance.

Gary Steck - Plans for the third 18 months: • Complete ITS-1 analysis for samples remaining from the isozyme study of Steck 1991 [Venezuela (highland and lowland), Brazil (Bahia, Sao Paulo), Costa Rica]; plus additional samples in the Florida State Collection of Arthropods from Ecuador, Peru, Bolivia, Brazil; plus colony samples from Seibersdorf (Brazil, Colombia, Mexico). (Collaboration with Bruce Sutton). • Facilitate the collection and establishment of a population from Peruvian highlands (and other areas if possible, e.g., Bolivia, Venezuela). (Collaboration with Bruce Sutton, Allen Norrbom, Erick Rodriguez) - Progress made up to 3 rd RCM: • Specimens from different regions in Peru and Bolivia were collected. • Preliminary ITS-1 sequence data indicates that A. fraterculus populations in Departamento Cusco (Andean highland and Amazonian mid-elevations) are clearly different from the Pacific coastal populations of Peru. • DNA sequences from the Cusco populations, as well as those from a mid-elevation population on the eastern slopes of the Andes in Bolivia, match those of the widespread Brazilian 1 morphotype.

CYTOLOGY Nelson Canal - 5 year plan: • Examine and compare karyotypes from different populations of A. fraterculus and A. obliqua from Colombia with previously described karyotypes. - Plans for the first 18 months: • Perform karyotype analysis of the Andean morphotype. - Plans for the second 18 months: • Perform karyotype analysis of the remaining A. fraterculus populations. - Plans for the third 18 months: • Describe the karyotype of the Mexican and Ecuadorian morphotypes (in collaboration with Vicente Hernández-Ortiz). 22

- Progress made up to 3 rd RCM: • Karyotype of the Andean population was described for 4 A. fraterculus and 2 A. obliqua populations. • The karyotypes of seven populations of A. fraterculus from Colombia (Andean Morphotype) and four populations of A. obliqua were described.

MORPHOLOGY Vicente Hernández-Ort iz / E. Arévalo / G. Palma / J. Martinez / J. Tigrero - 5 year plan: • Perform a comparative morphometric analysis of populations of the nominal species Anastrepha fraterculus (Wiedemann) throughout Mexico and Central America in order to describe the involved species. • Study the morphological variability of populations from Colombia, Ecuador and Peru, comparing samples from lowland Pacific coastal plain with those samples from highland Andean areas from those countries. • Describe the inter-population variability and morphotypes established in each geographic area and identify their distribution patterns. • Determine the usefulness of morphological characters to recognize and describe the species of the complex. - Plans for the first 18 months: • Collect A. fraterculus field samples. • Preserve and mount structures. • Carry out digital imaging. • Measure structures in 12 populations. - Plans for the second 18 months: • Obtain samples from some countries in Central America (Costa Rica and Honduras) and additional populations from the Colombian highlands. • Complete the morphometric analysis intitated in the first phase of the CRP. • Prepare a protocol for sample preparation to be sent to participants of a training course to be held in Brazil in 2012. • Give a one to two week training course on morphometrics and morphotype identification of A. fraterculus . • Start preliminary analysis on wing shape using geometric morphometry for Mexican, Andean and Peruvian morphotypes. - Plans for the third 18 months: • Provide taxonomic support on morphometrics for groups studying chemical ecology, DNA sequences, and larval studies. • Compile host plant lists for Mexican, Andean, and Peruvian morphotypes. • Prepare a manuscript describing the Mexican, Andean and Peruvian morphotypes as new species. - Progress made up to 3 rd RCM: • Samples from 6 populations from Ecuador,4 populations from Colombia and 2 populations from Mexico were obtained. • Up to 80% of the samples were preserved and mounted. • Digital imaging were performed on those slides. • Almost 50% of the samples were measured for morphometric analysis. • Samples from Colombia (10 populations) and Ecuador (8 populations) were obtained. • The morphometric analysis of the Colombian and Ecuadorian populations was initiated and completed. 23

• A protocol for sample preparation for the participants of a morphometrics training course was elaborated. • A one week training course on morphometrics and morphotype identification of A. fraterculus was given in Brazil in November 2012. • The analysis of wing shape using geometric morphometry for Mexican, Andean and Peruvian morphotypes was completed.

Nelson Canal - 5 year plan: • Describe egg and larval external morphology of A. fraterculus and A. obliqua from Colombia. • Study adult morphology of different populations from A. fraterculus and A. obliqua from Colombia. - Plans for the first 18 months: • Collect A. fraterculus field populations. • Establish laboratory colonies. • Perform morphological studies on immature and adult stages. - Plans for the second 18 months: • Continue collections of A. fraterculus and A. obliqua from Colombia. • Start a morphometric analysis in collaboration with Vicente Hernández-Ortíz. • Describe egg morphology of A. fraterculus and A. obliqua . - Plans for the third 18 months: • Complete morphometric analysis of A. obliqua females. • Descriptive and morphometric studies on 3 rd instar larvae of Mexican, Andean, and Peruvian morphotypes (with G. Steck). - Progress made up to 3 rd RCM: • Samples were obtained from 5 A. fraterculus and 4 A. obliqua populations from Colombia. • Efforts to established colonies from all populations were made and at the moment two populations from A. obliqua have been colonized. • Adults from all populations were measured and analysis is in progress. • The mouthparts of 3 rd instar larve from some populations were measured using light microscope. • Nine populations of A. fraterculus and 11 populations of A. obliqua from Colombia were acquired. • The morphometric analysis of A. fraterculus was completed in collaboration with Vicente Hernández-Ortíz. The morphometric analysis of A. obliqua is underway including also 1 population from Mexico and 1 population from Brazil. • Training was provided to M. Rosario Castaneda to analyze egg morphology of Colombian populations of A. fraterculus and A. obliqua. • The morphological study of the 3 rd instar larvae of A. fraterculus from Colombia was completed.

Gary Steck - 5 year plan: • Develop a strategy for sampling, acquiring host plant and geographic distribution data, rearing and breeding specimens for diverse studies of adults and immature stages, preservation, and vouchering of specimens. 24

• Assist in the acquisition of field collections, as needed, based on participation of various agencies throughout the Americas. • Examine and describe immature stages of various populations of the A. fraterculus complex in search of diagnostic characters to separate discrete species, as provided by cooperating teams. - Plans for the first 18 months: • Design a population sampling strategy for the A. fraterculus complex throughout its geographic range that will maximize the types of data that can be used for description of discrete species, and determining their geographic distributions, and associations with host plants. • Describe immature stages of various populations of the A. fraterculus complex (contingent on provision of specimens by cooperating teams of the CRP). - Plans for the second 18 months: • Continue with the SEM and optical microscope observations on 3 rd instar A. fraterculus larvae. • Continue collaboration with Dutra et al. on description of immature stages of various species of Anastrepha . • Submit a proposal to get fundings from the US Farm Bill to strengthen collection network. • Attempt to establish collaboration with people from Universidad Nacional in Cuzco, Peru in order to obtain A. fraterculus material to be used in the CRP. - Plans for the third 18 months: • Complete the larval descriptions for 3 rd instar larvae of Mexican, Andean, and Peruvian morphotypes (in collaboration with Nelson Canal). • Acquire new collections of Anastrepha spp. and populations of the A. fraterculus complex in Peru (also other countries if permits can be obtained) (in collaboration with Bruce Sutton, Allen Norrbom, Erick Rodriguez). - Progress made up to 3 rd RCM: • Exploratory trip to Peru in search of collaborators for A. fraterculus field collections. • Samples from 4 A. fraterculus colonies established at Seibersdof and one from Peru were obtained. • SEM observations carried out on 3 rd instar larvae from 5 A. fraterculus populations representing 3 morphotypes. • Description of immature stages of various species within the fraterculus group resulting in 3 publications. • Erick Rodriguez was trained in SEM and optical microscope observations on 3 rd instar larvae of Anastrepha spp. • Collaboration with Dutra et al. continued to describe immature stages of various species of Anastrepha and results were published (Dutra et al. 2012). • A proposal was submitted and 2 years of funding from the US Farm Bill was received to acquire new collections of Anastrepha larvae and adults to improve ability to identify larvae using morphological and molecular techniques. • Collaboration with entomologists at the Universidad Nacional San Antonio Abad Cuzco, Peru was established to obtain samples of Andean populations of Anastrepha spp . • Two collecting trips were made to Peru and ca. 37 spp. of Anastrepha were obtained along with larvae collections from 14 host plants. • A collecting trip was made to Panama to acquire samples of Anastrepha adults and larvae for morphological and molecular study. 25

BEHAVIOUR Iara Joachim-Bravo / Vanessa Dias / Kelly Roriz - 5 year plan: • Evaluate the level of reproductive isolation among populations from three Brazilian morphotypes through demographic parameters (fertility, sex ratio, and larva and pupa viability), sexual compatibility and competitiveness. • Describe the pre-mating behaviour pattern from the three Brazilian morphotypes. • Analyze comparatively the acoustical signals emitted during the courtship in each morphotype. - Plans for the first 18 months: • Collect infested fruit in the field from different regions in Brazil. • Establish laboratory colonies of two Brazilian morphotypes ( A. sp 1 aff fraterculus and A. sp 2 aff fracterculus ) from the flies collected in the field. • Initiate compatibility tests between the two established colonies of the two Brazilian morphotypes. • Initiate laboratory video recordings in order to characterize the courtship behaviour of the two established colonies of the two Brazilian morphotypes. • Perform a comparative analysis of the sounds emitted by the flies during the courtship behaviour from each of the two established colonies of the two Brazilian morphotypes. - Plans for the second 18 months: • Preserve specimens from the A. fraterculus colonies studied for vouchering and DNA extractions. • Perform morphometric analysis to determine the morphotype to which each A. fraterculus population belongs in collaboration with Vicente Hernández-Ortíz. • Continue field sampling with emphasis on obtaining A. sp 3 aff fracterculus . • Continue mating compatibility among the already established A. fraterculus colonies. • Continue male sexual behaviour description (lekking behaviour and video recordings). • Start a comparative analysis of the sounds emitted by A. fraterculus flies during the courtship behaviour. - Plans for the third 18 months: • Continue collections at Una (BA, Brazil northeast region), Parnamirim (RN, Brazil northeast region), Alagoas (in collaboration with Ruth do Nascimento in the northeast region). • Establish new laboratory colonies of A. fraterculus from the flies collected in the field to be used in compatibility and behavioural tests. • Publish compatibility and behavioural tests finished among the southern and southeast populations. • Continue the compatibility tests comparing populations from northeast, southeast and southern regions of Brazil established at laboratory as follows: Parnamirim x Bento Gonçalves (new replicates) and Parnamirim x Piracicaba (to be done once the morphotype identification is finished). • Continue laboratory video recordings tests to characterize the courtship behaviour of different populations (Parnamirim, Bento Gonçalves and Piracicaba). • Continue the analysis of acoustical communication during mating for the Parnamirim population. • Continue post-zygotic compatibility tests with the combinations of populations: Parnamirim x Bento Gonçalves; Parnamirim x Piracicaba and Bento Gonçalves x Piracicaba. 26

- Progress made up to 3 rd RCM: • Collections were performed of 6 A. fraterculus populations from south Brazil, 1 from the Southeast and 7 from the Northeast (morphotypes to be defined). • Laboratory colonies were established resulting in 6 A. fraterculus colonies from the south, 1 from southeast and 1 from the northeast. • Mating compatibility evaluations were performed among 6 of those A. fraterculus populations (Bento Gonçalves, Pelotas, Vacaria, Sao Joaquim, Piracicaba and Natal (morphotypes to be defined). • The lekking behaviour was studied for 3 A. fraterculus populations from the south and 1 from the southeast. • Video recordings of male courtship were obtained for one A. fraterculus population from each region. • Mating compatibility evaluations among four populations from southern of Brazil (Bento Gonçalves, Pelotas, Vacaria and São Joaquim) were completed. • Mating compatibility tests between a southeast population from Brazil (Piracicaba) and southern populations (Vacaria; São Joaquim and Bento Gonçalves) were completed. • Mating compatibility tests between Parnamirim (northeast) vs. Piracicaba (southeast) and Bento Gonçalves (south) populations were initiated. • The courtship behaviour analysis of Bento Gonçalves, Piracicaba and Parnamirim populations were initiated. • The sound analysis of south and southeast populations was completed. • Kelly Roriz participated in the morphometric course conducted by Vicente Hernández-Ortiz in 2012.

Teresa Vera / Diego Segura / Solana Abraham / Juan Rull - 5 year plan: • Perform mating compatibility tests from as many A. fraterculus morphotypes as possible. • Characterize aspects of the sexual behaviour of each population. • Determine the occurrence of post-zygotic isolation among populations. • Study the sexual behaviour of the hybrids. - Plans for the first 18 months: • Establish / renew A. fraterculus colonies from the putative species at Seibersdorf. • Perform mating compatibility tests among the colonies. • Perform post-copulatory behaviour studies. • Perform post-zygotic isolation studies among the different A. fraterculus colonies. - Plans for the second 18 months: • Preserve specimens from the A. fraterculus colonies for vouchering and DNA extractions. • Establish new A. fraterculus colonies in Seibersdorf with emphasis on the Andean morphptype (Colombia), A. sp 2 aff fracterculus (colony from Maceio if Vicente Hernández-Ortíz confirms its identity), the Mexican morphotype and the Peruvian morphotype. • Continue mating compatibility studies with the A. fraterculus colonies established at Seibersdorf in collaboration with Nelson Canal. • Perform experiments in field cages in Seibersdorf with artificial leks to determine if females show different attraction to the pheromones of males from the different morphotypes. 27

• Continue female post-copulatory behaviour studies. • Obtain extracts from the male accessory glands in order to characterize the proteins. - Plans for the third 18 months: • Perform experiments in field cages in Seibersdorf with artificial leks to determine if females show different attraction to the pheromones of males from those morphotypes/populations that exhibit the same time of mating activity but some degree of sexual isolation (Brazilian 1 and Mexican morphotype and Piracicaba). • Perform bioessays with pheromone extracts to determine female attraction to the pheromones of males from other morphotypes (in collaboration with Blanka Kalinova, Radka Brizova and Peter Teal). • Obtain extracts from the male accessory glands in order to determine female receptivity after the injection of the extracts. • Initiate the description of the protein profile of the male accessory glands’ proteins (in collaboration with Seibersdorf and INBIOTECA, Mexico). - Progress made up to 3 rd RCM: • Four new A. fraterculus colonies were established at Seibersdorf (Mexico, Vacaria, Pelotas and Piricaba). • Mating compatibility between the Mexican morphotype and the Peruvian morphotype, as well as between the Mexican morphotype and two populations from Brazil (Vacaria and Pelotas) and one population from Argentina, was evaluated. • Female post-copulatory behaviour of the Peruvian morphtype and one population from Argentina, after mating with males from the same origin or with males from the other population, were evaluated. • Post-zygotic incompatibility between one population from Argentina (Tucumán) and two populations from Brazil (Vacaria and Pelotas) was evaluated. • Preliminary experiments to evalauate post-zygotic incompatibility between the Mexican morphotype and the Peruvian morphotype, and between the Mexican morphotype and a population from Brazil, were performed. • New A. fraterculus colonies were established at Seibersdorf (Perú, Colombia, Mexico and Parnamirim). • Mating compatibility between the Colombian morphotype was evaluated with the Peruvian, Mexican and A. ff morphotype. • The evaluation of female post-copulatory behaviour of the Peruvian morphtype and one population from Argentina after mating with males from the same origin or with males from the other population was completed and the manuscript was sent for publication. • Specimens from the A. fraterculus colonies for vouchering and DNA extractions were preserved and distributed as agreed (in collaboration with Norma Nolasco and María del Rosario Castañeda).

CHEMICAL ECOLOGY Ruth do Nascimento / Blanka Kalinova / Lucie Vanickova / Radka Brizova - 5 year plan: • Compare the exact composition of the volatile pheromone profile released by A. fraterculus males from different morphotypes. • Compare the exact composition of the cuticular hydrocarbons (CHCs) profile of A. fraterculus males from different morphotypes. 28

• Study the CHCs and cuticular esther differences between males and females from different ages to improve understanding of pre-zygotic isolation. • Perform behavioural assays to identify the biological relevance of pheromone differences between populations. - Plans for the first 18 months: • Collect and establish A. fraterculus populations under laboratory conditions from two morphotypes ( A. sp 1 aff. fraterculus and A. sp 2 aff. fraterculus ). • Collect and characterize volatiles from calling males of the two A. fraterculus morphotypes. • Extract and characterize cuticular hydrocarbons from males and females of the two A. fraterculus morphotypes. • Perform Gas chromatography-Electroantennography (GC-EAG) analyses of male extracts. - Plans for the second 18 months: • Preserve specimens from the A. fraterculus colonies for vouchering and DNA extractions. • Perform morphometric analysis to determine the morphotype to which each population belongs in collaboration with Vicente Hernández-Ort íz. • Identify chemically the volatiles from the remaining populations by GC-MS and GC- GCMS. • Initiate gas chromatography-electroantennography (GC-EAG) analyses of male extracts in case that behavioural data provide evidence of differences in female behaviour. - Plans for the third 18 months: • Collect field infested guavas in the states of Alagoas and Parnamirim (Rio Grande do Norte). • Establish cultures of these populations under laboratory conditions. • Maintain the A. fraterculus populations derived from Alagoas, Parnamirim, Bento Gonçalves, Piracicaba, São Joaquim, Pelotas and Vacaria under laboratory conditions. • Complete the identification of the morphotypes (in collaboration with other Brazilian participants). • Perform the experiments described below in order to obtain enough extracts for GC x GC/TOF-MS analyses of all the populations collected (Alagoas, Parnamirin, Bento Gonçalves, Piracicaba, São Joaquim, Pelotas and Vacaria) aiming to: a) identify and quantify the pheromone components and cuticular hydrocarbons of those populations which have not yet being studied; b) conduct laboratory bioassays to find out which role they play in the communication among conspecific and heterospecific males and females; • Carry on electrophysiological studies to find out which compounds within the mixtures of volatiles released by four populations of A. fraterculus , from three different regions of Brazil: Northeast: Alagoas; South: Bento Gonçalves and São Joaquim; Southeast: Piracicaba, trigger a response on the antennae of conspecific and heterospecific females. These analyses will be performed in Brazil. • Use statistical tools to find out similarities and differences between them. • Analyse the pheromone of a Colombian population (collected by Nelson Canal) by GCxGCMS in Prague. • Perform GC-EAD for the available morphotypes different than Brazilian 1 (to be done in Prague). - Progress made up to 3 rd RCM: 29

• Laboratory A. fraterculus colonies were established resulting in 4 colonies from the south, 1 from southeast and 2 from the northeast from Brazil (morphotypes to be defined). • Pheromone volatiles from two of these A. fraterculus populations (Bento Gonçalves and Maceio belong probably to two morphotypes (morphotypes to be defined) were collected and identified. • The cuticular hydrocarbons from these two A. fraterculus populations were also analysed. • The pheromone volatiles from several populations involving at least two morphotypes were characterized (Peruvian and Brazilian 1 morphotypes). • The chemical characterization of the pheromone volatiles from another two morphotypes was initiated (Mexican and Andean morphotypes). • The cuticular hydrocarbons from several populations comprising the four above mentioned morphotypes were characterized.

Teresa Vera / Diego Segura / Solana Abraham / Juan Rull - 5 year plan: • Characterize the male sexual pheromone profile of each A. fraterculus morphotype. - For the first 18 months: • Collect male sexual pheromone from A. fraterculus colonies established at Seibersdorf, Austria. - Plans for the second 18 months: • Collect pheromone and CHC samples from the Andean and Mexican A. fraterculus morphotype. • Characterize the male sexual pheromone and CHC profile of the samples collected in collaboration with Blanka Kalinova and Peter Teal. - Plans for the third 18 months: • Extract pheromones in co-operation between Seibersdorf and Prague to be used in the cross-attraction bioassays. • Check pheromone quality (in collaboration with Blanka Kalinova, Radka Brizova and Peter Teal). - Progress made up to 3 rd RCM: • Male sexual pheromone from 5 A. fraterculus colonies established at Seibersdorf, Austria was collected. • The volatiles as well as the CHCs from several populations from Seibersdorf were collected and sent to Prague and Gainesville for chemical characterization.

Bactrocera cucurbitae

Background Situation Analysis

Bactrocera cucurbitae is a major pest of cucurbit crops and has spread from its area of origin (South East Asia) across Africa, Hawaii, the Indian Ocean, Papua New Guinea and the Solomon Islands (Severin et al ., 1914; Dhillon et al ., 2005). In particular, it causes severe losses in food crops in poor village communities and restrictions to trade in some cucurbit crops.

Some populations have been identified in Africa, Indian Ocean, Hawaii and South East Asia that indicate that very closely related species may be present. This problem needs to be resolved before species-specific treatments such as the SIT can be effectively applied in all situations and regions. There are a number of single species that differ in host, morphology, etc. from region to region and B. cucurbitae may be one of these.

Recent population genetic studies can resolve five major groups worldwide (with microsatellites): African mainland + Seychelles, Réunion + Mauritius, Central Asia, SE Asia, Hawaii (Virgilio et al. , 2010). However phylogeographic patterns could not be discerned (using mitochondrial and nuclear gene fragments (total of 2764 bp) Virgilio, unpublished).

Field cage compatibility tests were recently conducted between populations of Mauritius, Seychelles and genetic sexing strain of Hawaii. No incompatibility was found (Sookar et al. , 2010). Further mating tests could help to resolve their species status.

Baseline and Progress on Bactrocera cucurbitae

DNA - Knowledge at the start of the CRP: • General information on phylogeography / population genetics available on a worldwide basis. Also information available on population genetics with regard to the African mainland and La Réunion. - Gaps identified: • More data on potential host races are needed, both within Cucurbitaceae (cultivated versus wild) and cucurbits versus non-cucurbits. - Progress up to 3 rd RCM in addressing identified gaps: • Detailed DNA studies on population genetics in Réunion, and to a lesser extent in Tanzania, have shown that there is no relation between genetic structure and host plants (i.e. cultivated versus wild cucurbits). • Mitochondrial variation in B. cucurbitae populations from Hawaii were studied and little variation detected. Other (nuclear) markers and populations are currently being explored.

CYTOLOGY - Knowledge at the start of the CRP: • Work has been carried on two B. cucurbitae populations regarding karyotyping and polytene maps. - Gaps identified: • Need to have same information from other populations.

- Progress up to 3 rd RCM in addressing identified gaps: • No cytogenetic differences found between two populations of B. cucurbitae (Genetic Sexing Strain Hawaii and Bangladesh wild type) (Zacharopoulou et al. , 2011). • Karyotyping reveals that B. cucurbitae is significantly different from other Bactrocera (i.e. position of subgenus Zeugodacus in relation to Bactrocera and Dacus ).

ALLOZYMES - Knowledge at the start of the CRP: • Previous work done by Malacrida et al. (1996). - Gaps identified: • No further studies planned; covered by DNA work.

GENOMICS - Knowledge at the start of the CRP: • No studies conducted. - Gaps identified: • Need to conduct genomic analysis. - Progress up to 3 rd RCM in addressing identified gaps: • No further studies conducted.

MORPHOLOGY - Knowledge at the start of the CRP: • B. cucurbitae can be morphologically differentiated from other species within the subgenus Zeugodacus . - Gaps identified: • Possible confusion with other valid taxa. • Some populations appear to indicate that very closely related species may be present. - Progress up to 3 rd RCM in addressing identified gaps: • No other valid taxa have been identified that could cause possible confusion with B. cucurbitae . Also no differences identified among populations. Therefore no need for further studies.

MORPHOMETRICS - Knowledge at the start of the CRP: • Unexplored so far. - Gaps identified: • Could provide additional information for recognition of potential geographic or host races. Only preliminary work done at Seibersdorf for testing methodology. - Progress up to 3 rd RCM in addressing identified gaps: • No studies conducted so far.

MALE LURE RESPONSE - Knowledge at the start of the CRP: • Attraction of males to cue lure and melolure, not by methyl-eugenol. - Gaps identified: • No need for further study with regard to the CRP.

DEVELOPMENTAL PHYSIOLOGY - Knowledge at the start of the CRP: 32

• Data are available from Japan, Hawaii and La Réunion, but have not yet been compared. - Gaps identified: • Need to compare existing biological data from countries where host range is different. - Progress up to 3 rd RCM in addressing identified gaps: • No studies conducted so far.

BEHAVIOUR - Knowledge at the start of the CRP: • Good knowledge of mating behaviour through studies in Japan, Hawaii and recently in Seibersdorf. - Gaps identified: • Mating compatibility in future if need arises. - Progress up to 3 rd RCM in addressing identified gaps: • Publication of compatibility studies (Sookar et al. 2010), investigating cross-mating among three populations of B. cucurbitae (Hawaii, Seychelles and Mauritius). No sexual isolation was discovered.

PHEROMONE COMPONENTS AND CONCENTRATIONS - Knowledge at the start of the CRP: • Previous work done by Japanese researchers. - Gaps identified: • No need for further work planned in the immediate future; perhaps at later stage to relate pheromones with potential host races. - Progress up to 3 rd RCM in addressing identified gaps: • Pheromone analysis and profile on-going for Genetic Sexing Strain from Hawaii: effect of age and diet (Haq et al ., in prep).

HOST RANGE - Knowledge at the start of the CRP: • Well documented as a whole. - Gaps identified: • See comment under DNA: possibility of host races. - Progress up to 3 rd RCM in addressing identified gaps: • Work by Sookar on host preference; host range studied specifically in Tanzania (Mwatawala et al. 2010). Host range in Réunion and Tanzania was studied in relation to genetic structure. • Laboratory experiments on behaviour and preimaginal development of populations from Hawaii and Mauritius on cucurbit and non-cucurbit hosts showed no differences in egg laying choice and survival of larvae to adults. • Kenya: (Ekesi). In the studies involving the host range of B. cucurbitae , the insect was reared from a total collection of 17 plant species comprising 10 families covering Cucurbitaceae, Solanaceae, Anacardiaceae, Rutaceae and Myrtaceae from surveys carried out at the Coast, Eastern and Central Provinces of Kenya. • Populations of B. cucurbitae vary in their preference to host plants and this preference is driven also by variation among population of the pest. • Two populations were established originating from bitter gourd and tomato. In the bitter gourd population, significantly higher number of puparia was recovered from 33

bitter gourd but other cucurbits were also infested. The tomato population exclusively preferred tomato compared to the other host plants. • Tanzania: a study on host preference studies under natural infestation and under semi- natural conditions (cages) are being conducted, testing three hosts; cucumber, pumpkin and watermelon. Work is in progress.

CUTICULAR HYDROCARBONS - Knowledge at the start of the CRP: • No studies yet specifically with regard to B. cucurbitae cuticular hydrocarbons have been conducted. - Gaps identified: • Many gaps, however, no need for work in the immediate future, unless there is a need to relate cuticular hydrocarbons with potential host races. - Progress up to 3 rd RCM in addressing identified gaps: • General methodology has been developed for Tephritidae to analyse cuticular hydrocarbons. • The CHCs from males and females of B. cucurbitae - age dependent production (day 5, 15, 20, 30 after emergence) were extracted. The extracts were analysed by GCxGCMS. The preliminary data demonstrate sex-specific differences.

Specific Progress and Individual Research Plans: Bactrocera cucurbitae

TAXONOMY Marc de Meyer - 5 year plan: • Provide morphometric diagnostic markers to differentiate potential geographic and/or host races. - Plans for the last 18 months: • Considering outcome of genetic studies on potential host races, no further activities are required at this stage. - Progress made up to 3 rd RCM: • Not done so far; only in case of identification of host races.

Sunday Ekesi / Fathiya M. Khamis - 5 year plan: • Collect and share biological material of B. cucurbitae with other CRP participants as required . - Plans for the last 18 months: • Make material available for other CRP studies when required. - Progress made up to 3 rd RCM: • Two colonies (one population from bitter gourd and one from tomato) have been established at ICIPE.

BEHAVIOUR / HOST RANGE Hélène Delatte and Serge Quilici - 5 year plan: • Define the host range of La Réunion populations through behavioural experiments (choice and non-choice on mango and solanaceous fruits) including interspecific competition with other fruit flies. 34

• If required (based on results from Hélène Delatte, Serge Quilici and Maulid Mwatawala): test mating incompatibility between geographic and/or potential host races at Seibersdorf. - Plans for the last 18 months: • Continue behavioural studies in olfactometers. - Progress made up to 3 rd RCM: • Ecological studies in La Réunion allow specifying the host range of B. cucurbitae among wild and cultivated cucurbits at various sites and altitudes. • Study of B. cucurbitae intra- and interspecific competition was conducted and finalized. • Behavioural studies in olfactometers and field cages to specify the preferences of female B. cucurbitae for different species of host plants are under progress.

Maulid Mwatawala - 5 year plan: • Host plant preferences, incidence and infestation rates of B. cucurbitae in Tanzania. Verification of host choice and the relationship with intraspecific variation. - Plans for the last 18 months: • Continue conducting host preference (cucurbit versus non-cucurbits) tests in semi- natural conditions. • Sending material from non-cucurbit hosts to CIRAD and RMCA for genetic analysis. - Progress made up to 3 rd RCM: • Surveys were conducted throughout Tanzania along several transects, obtaining material from cucurbit and non-cucurbit hosts. • Material from cucurbit hosts (wild and cultivated) were sent to Royal Museum CA for genetic analysis. • Tanzania. Host preference of B. cucurbitae under natural and semi-natural infestation is being tested for three hosts (cucumber, pumpkin and water melon).

Sunday Ekesi - 5 year plan: • Host plant preferences, incidence and infestation rates of B. cucurbitae in Kenya. Verification of host choice and the relationship with intraspecific variation. - Progress made up to 3 rd RCM: • In the studies involving the host range of B. cucurbitae , the insect was reared from a total collection of 17 plant species comprising 10 families covering Cucurbitaceae, Solanaceae, Anarcadiaceae, Rutaceae and Myrtaceae from surveys carried out at the Coast, Eastern and Central Provinces of Kenya. • Two populations were established originating from bitter gourd and tomato and differences in preference were observed.

MOLECULAR / CYTOGENETICS Antigone Zacharopoulou / Antonios Augustinos / Farzana Yesmin - 5 year plan: • Confirmation karyotype and polytene similarity among the different geographic B. cucurbitae populations. - Plans for the last 18 months: • Pending outcome of genetic studies on potential host races, no further activities are planned at this stage. 35

- Progress made up to 3 rd RCM: • Wild populations of GSS Hawaii and Bangladesh were studied. Karyotyping reveals that B. cucurbitae is significantly different from other Bactrocera (i.e. position of subgenus Zeugodacus in relation to Bactrocera and Dacus ). • There are no cytogenetic differences between the populations of B. cucurbitae studied.

Massimiliano Virgilio and Hélène Delatte / Serge Quilici / Dave Haymer - 5 year plan: • Analysis of the population structure according to potential host races in Tanzania and La Réunion. - Plans for the last 18 months: • Given earlier results, the use of microsatellites to differentiate potential host races within B. cucurbitae will not be further investigated. • Use the B. cucurbitae transcriptome dataset to identify genes and gene products in biological significant pathways (odorant and pheromone binding proteins; carbohydrate metabolism). This will be explored by the University of Hawaii at Manoa to provide complementary information on the existence of host races. - Progress made up to 3 rd RCM: • Genetic structure for different African populations, including La Réunion was analysed. Weak genetic structuring was found on a continental scale. • Detailed studies on population genetics in La Réunion; three groups were recovered but there was no relation between the genetic structure and host plants (i.e. cultivated versus wild cucurbits). • A smaller pilot study in Tanzania showed no obvious relationship between hosts and genetic structure. • Mitochondrial variation in Hawaiian populations is very little.

DEVELOPMENTAL PHYSIOLOGY Marc De Meyer - 5 year plan: • Literature review: comparison of biological data on developmental rates between polyphagous (Hawaii) and oligophagous (Mascarenes) strains of B. cucurbitae. - Plans for the last 18 months: • Conduct literature review study on B. cucurbitae developmental physiology. - Progress made up to 3 rd RCM: • So far no studies conducted.

PHEROMONE COMPONENTS AND CUTICULAR HYDROCARBON Peter Teal / Lucie Vanickova / Sunday Ekesi / Maulid Mwatawala - 5 year plan: • Pheromone analysis and profile for Genetic Sexing Strain from Hawaii: effect of age and diet. - Plans for the last 18 months: • Assess pheromones and cuticular hydrocarbons released by two populations from different host plants. Assess chemical host plant preferences from two populations from different host plants. - Progress made up to 3 rd RCM: • The CHCs from males and females of B. cucurbitae - age dependent production (day 5, 15, 20, 30 after emergence) were extracted. The extracts were analysed by GCxGCMS and species specific differences were found. 36

Bactrocera dorsalis Complex

Background Situation Analysis Across Asia and the Pacific the fruit fly subfamily Dacinae contains some 48 recognised pest species (Drew, pers. comm.). Of these 48 pest species, eight are currently recognized within the Bactrocera dorsalis complex with some being the worst of all pest species within the subfamily (Drew and Hancock, 1994). Known collectively as the Oriental fruit fly complex, they are also categorized as the fourth worst of all global insect pests. While it is difficult to put a precise value on the economic losses, these have been estimated by the Griffith University International Fruit Fly Centre in Australia to be approximately US$1 billion per annum for the Asian region. These losses include destruction of crops, restriction of international trade, and the establishment of a range of quarantine and regulatory activities carried out by various regional governments.

Background research has generated data on diagnostics, field surveillance including quarantine strategies, field pest control, and some export trade (e.g. Tan and Nishida, 1996, 1998; Muraji and Nakahara, 2002; Naeole and Haymer, 2003; Smith et al ., 2003; Armstrong & Ball, 2005; Vargas et al 2009). This knowledge has also identified key gaps in resolving the B. dorsalis complex, but there is no consensus on species limits of the major Bactrocera dorsalis complex pest species, particularly B. dorsalis s.s. , B. papayae , B. philippinensis , B. carambolae and potentially B. invadens (Clarke et al ., 2005; Drew et al ., 2008; Wee and Tan, 2005; Ebina and Ohto, 2006). Failure to resolve this complex prevents further development towards SIT implementation in Area-Wide Integrated Pest Management (AW-IPM) programmes against these pest insects and limits international trade.

The Asian region has been a primary source of invasions of species of the B. dorsalis complex into many other regions of the world, including Africa, Middle East, northern and southern Pacific Islands (including Papua New Guinea (PNG) and Australia), Indian Ocean, and South America. Currently some of these introduced species are causing food security and market access problems in Africa, PNG, and Pacific Islands. Background research into the taxonomy of the B. dorsalis complex has been unable to provide definitive identification of some species (Clarke et al ., 2005). This confounds the collection of associated host plant records and geographic distributions. It is absolutely essential that the species be accurately identified in order to be able to apply AW-IPM field programmes that include an SIT component.

Because trade implications and response systems to detections and/or incursions are not the same for all species in the complex, "near-enough" identification is not, unfortunately, good- enough. Consequently Member States have difficulty overcoming the phytosanitary barriers to export trade that satisfy major importers such as Japan, South Africa, USA, Australia, and New Zealand. Not least is the severe problem arising if one member of the complex is detected or becomes established in a Member State but is unable to be differentiated from others in the complex. In this case that State would then be forced to indicate that any and all members of the complex may in fact be present, which would result in trade embargoes.

In this component of the CRP, a comprehensive biological, morphological, chemo-ecological and molecular study on pest species of the B. dorsalis complex will be undertaken so as to:

(i) Resolve species limits by seeking a consensus result from different tests including: 37

(a) Identifying the levels of inter- and intra-specific genetic ( e.g. cytology) and molecular variation, (b) Carrying out morphological and morphometric analysis, (c) Carrying out biological tests including cross-mating and host use studies, (d) Pheromone analyses to compare component ratios across putative species (ii) Examine congruence between data from above mentioned studies, (a) to (d) which may support taxonomic revision or retain existing species, and (iii) Develop robust diagnostic tools for the resolved species.

Baseline Knowledge on Five Priority Species in the Bactrocera dorsalis Complex

DNA - Knowledge at the start of the CRP: • Mitochondrial DNA o COI ‘barcode’ gene region, and other mtDNA markers, show no clear distinction between currently defined species; data from more populations may improve this technique for population/species differentiation. • Nuclear ribosomal DNA o ITS1+2 diagnostic for separating B. carambolae from remaining four species. • Microsatellites o Sequences from B. dorsalis s.s ., B. invadens , B. oleae and B. papayae are available and may be useful for population genetic structure but not sure if they will work with the B. dorsalis complex for species separation. • Nuclear coding gene data for 16 loci (Muscle-specific actin, guanylyl cyclase receptor, vitellogenin 1&2 precursors, 70 kDa heat shock protein, white, cytochrome P450, guanylyl cyclase receptor beta, beta-2 tubulin gene, acetylcholinesterase, odorant receptor, oderant binding, chemosensory, doublesex, G6PDH, alcohol dehydrogenase), all of which for B. dorsalis s.s. ; only two of these involve other species in the complex and include at least B. carambolae, B. papayae and B. philippinensis , but species differentiation has not been assessed. • Exon primed intron crossing from actin gene – for B. papayae and B. carambolae and B. dorsalis s.s. showed distinction but population level analyses have not been undertaken. - Gaps identified at the start of the CRP: • There is no adequate and consistent sample coverage for the five target species. • The lack of discriminatory characters means that either a) the characters exist but are yet to be discovered, or b) that such characters do not exist and the species are the same. This highlights the question as to how many negatives do we accept before stopping? • The inferences from current knowledge are based on standard phylogenetic and phylogeographic analyses which may be a limitation to accurate interpretation. - Progress up to 3 rd RCM in addressing identified gaps: • Significantly improved sample coverage, including from China, Indian subcontinent, Myanmar, Indo/Malay Archipelago, and African populations. While geographic coverage has increased, the numbers of individuals from particular locations may still be insufficient. • Additional diagnostic genetic markers (mitochondrial, ribosomal and nuclear genes) have been developed using a comparative transcriptome approach, but they do not discriminate between four of the five species; B. carambolae forms a monophyletic 38

group according to COI and ITS1 supporting its current species-level status. Bactrocera kandiensis is also a well resolved clade. • A full multigene phylogenetic analysis has been completed. • Demography (using microsatellites) of B. invadens has been published (Khamis et al 2008) and that for B. dorsalis s.s. has been completed (indicating origin of the species is China). • Additional experiments have been undertaken to supplement the phylogenetic/phylogeographic analyses towards clarifying species limits, including mating compatibility, cytological, genomic, pheromone studies, morphometric and morphological studies. • Markers have been developed for the Y chromosome in B. dorsalis s.s. from Thailand. Differences have so far been found between B. dorsalis and B. carambolae , but not for among B. dorsalis, B. papayae and B. philippinensis (but sample sizes to be increased).

CYTOLOGY - Knowledge at the start of the CRP: • Studies on mitotic karyotypes identified several forms within the B. dorsalis complex, but did not definitively distinguish between putative species (e.g. Baimai et al ., 2000 ). - Gaps identified at the start of the CRP: • Further polytene maps are needed to allow distinguishing between putative species. • Polytene mapping for these species could possibly be linked with genomic data. - Progress made up to 3rd RCM in addressing identified gaps: • Polytene maps are now available for B. dorsalis s.s. (Zacharopoulou et al ., 2011). • For B. papayae salivary gland polytene chromosome maps, metaphase karyotypes and their morphometric characteristics have been described. In the case of B. carambolae , metaphase karyotypes and their morphometric characteristics have been described with construction of polytene chromosome maps in progress. • There are no structural differences between all five species regarding polytene chromosome banding patterns. • Hybrid analysis ( B. dorsalis s.s x B. invadens and B. dorsalis s.s. x B. carambolae bidirectional hybrid) also shows no chromosomal structural differences between paternal polytene chromosome banding pattern or their F1 hybrid, since hybrids’ polytene chromosomes are tightly synapsed. Minor asynapses were observed in a few hybrids between B. dorsalis and B. carambolae . • Localization of six unique sequences to polytene chromosomes ( in situ hybridization) has started for the three members of the complex and their hybrids (as in previous point) with localization of the hsp70, GLD, OVO, TRA, SXL, Scarlet genes.

ALLOZYMES - Knowledge at the start of the CRP: • Not applicable as a means to discriminate between species as it is too dependant on climatic factors. • Thai team tried allozymes several years ago but data are not published. - Gaps identified at the start of the CRP: • Considered not as priority technique for this CRP. - Progress made up to 3rd RCM in addressing identified gaps: 39

• Preliminary esterase isozyme studies using polyacrylamide gel and laboratory colonies of B. papayae and B. carambolae have been completed. This can differentiate between these species in the adult stage.

GENOMICS - Knowledge at the start of the CRP: • piggyBac and other mobile elements (in particular the Hopper element) have been documented to be present in some B. dorsalis species populations and may be used for species differentiation. Noted as difficult to apply for defining species limits. • An unpublished Hawaiian B. dorsalis s.s. genome exists. • Also an unpublished C. capitata genome which could be used as a reference. • Transcriptomics – under way for B. dorsalis s.s ., B. philippinensis and B. carambolae . There are potentially some markers for these species within that data set. - Gaps identified at the start of the CRP: • Current unavailability of the genome information. - Progress made up to 3rd RCM in addressing identified gaps: • piggybac is multiple copy, a characteristic which has been shown to differentiate B. dorsalis s.s. and B. invadens . • There is now a public web portal for accessing the current scaffold and contig structure of the Hawaiian B. dorsalis s.s. genome. Annotation is in progress. Whole body transcriptome data has been published for B. dorsalis s.s. (Taiwan, base line lab strain, 5-day old). Whole body transcriptome data developed for B. dorsalis s.s. (Saraburi, Thailand, F2-3) has been provided to the Hawaiian laboratory (USDA- ARS-PBARC, Hilo) to assist with annotation and placement of introns that may be useful for species marker development. • The genome sequence for C. capitata as a potential reference is under development. • Transcriptomics for different tissues and the two sexes are available for C. capitata and deposited in Vectorbase (Genbank). This will be used to help interpret the genomic data being developed for both C. capitata and B. dorsalis as well as to interpret the whole body transcriptome data that has been developed for B. dorsalis s.s. , B. papayae, B. philippinensis, B. carambolae and B. invadens . These data have been used to identify sequences/genes related to odorant- and pheromone-binding proteins, reproductive behaviour/reproduction, mate and host recognition. • Comparative transcriptome data for B. dorsalis s.s. , B. papayae, B. philippinensis, B. carambolae and B. invadens have been analysed for ‘species’ specific SNPs. • Y-chromosome sequence data have been developed for C. capitata and is under development for B. dorsalis s.s. These data can be used for sexing individuals at any stage of development.

MORPHOLOGY - Knowledge at the start of the CRP: • A revision of the SE Asian Dacini is under way, but is not expected to impact on the four nominated pest species in this CRP (Drew, pers. comm.). - Gaps identified at the start of the CRP: • Egg and immature morphology – not investigated for B. dorsalis complex species. • Explicit intra-specific population-level variation in both external and internal morphological characters. • Insufficient understanding of relative environmental/genetic influences on morphological phenotype. 40

- Progress made up to 3rd RCM in addressing identified gaps: • Major revision of SE Asian tephritids is complete. Updated information on all five species included. The status of B. philippinensis will change. • B. invadens vs B. dorsalis s.s. colour morphs described. • Egg and immature material from Seibersdorf lab colonies has been gathered and is being included as part of a major project describing the immature stages of tephritids. • The range of morphological variation has been assessed for B. dorsalis s.s and B. invadens from across their geographic ranges. • Morphological pattern variation in B. dorsalis s.s (Seibersdorf culture) has been assessed for flies fed varying quantities of food (standard lab diet). No clear correlation was observed. • Crosses have been undertaken between colour morphs (pale brown and black scutum) of B. dorsalis from Pakistan. Preliminary results suggest colour morph is a simple qualitative genetic trait.

MORPHOMETRICS - Knowledge at the start of the CRP: • A relatively large number of papers on morphometrics for B. dorsalis complex species exists. In some cases resolution occurs but not always. Often also with overlap between morphometric traits and often impossible to determine what might be within and between population variation. • Geometric morphometrics (shape analysis) shows some promise but needs further research. • Morphometric approaches have rarely been linked adequately with other morphological or genetic approaches. - Gaps identified at the start of the CRP: • Explicit intra-specific population-level variation in both external and internal morphometric characters. • Insufficient understanding of relative environmental/genetic influences on morphometric phenotype. - Progress made up to 3rd RCM in addressing identified gaps: • Aedeagi have been measured from 200+ specimens of B. dorsalis s.s ., B. carambolae, B. papayae, B. invadens (and B. verbascifoliae ) from SE Asia and Africa. The same samples have been sequenced for the COI gene and microsats and the combined dataset has been analysed. Bactrocera papayae aedeagi are longer as expected ( B. verbascifoliae is shorter, but they are also smaller flies). • Aedeagi have been measured from a latitudinal gradient from northern Thailand to Peninsular Malaysia for B. dorsalis s.s . and B. papayae. Results show a significant and continuous latitudinal cline from north to south, with northern Thailand flies the shortest and Malaysian flies possessing the longest aedeagi. • Morphometric analysis using wing veins and tibia lengths was also used to discriminate B. invadens from different localities, and with comparison with other Bactrocera species ( B. kandiensis, B. dorsalis s.s ., B. zonata, B. correcta, B. cucurbitae and B. oleae ). The results showed no resolution for species within the B. dorsalis complex (but they did separate from the non-dorsalis flies) and these results were also reflected in the COI sequence analysis. • Geometric morphometric wing shape data has been obtained for B. dorsalis s.s ., B. papayae, B. carambolae, B. philippinensis, B. occipitalis, B. cacuminata, B. opiliae, and B. musae. Results are consistent with B. dorsalis s.s ., B. papayae and B. 41

philippinensis representing the same species which displays strong isolation-by- distance patterns within SE Asia. All ‘outgroup’ species ( B. occipitalis, B. cacuminata, B. opiliae and B. musae ) resolve strongly from the four target species studied here. • Bactrocera invadens morphometric data has been analysed for aedeagus length, postsuttural lateral vittae and wing shape. This has been compared with B. dorsalis from across its geographic distribution and reveals no evidence of species-level differences. • Morphometrics of genitalia, head width and wings have been undertaken for B. dorsalis s.s . (Seibersdorf colony) reared under different larval densities. Eggs of a range of species have been measured. • Juvenile stages of all target species have been collected into alcohol and passed to G. Steck for morphological examination.

DEVELOPMENTAL PHYSIOLOGY - Knowledge at the start of the CRP: • There are differences between B. carambolae and B. papayae and B. dorsalis s.s. e.g. in attaining sexual maturity and in age-related development of response to parapheromones – e.g. work done at Seibersdorf and by Dr Tan on B. carambolae (Wee & Tan 2000; Wee et al . 2002; Wee & Tan, 2007). • There are differences in ovariole numbers between some Bactrocera species but has not been tested on B. dorsalis complex species. - Gaps identified at the start of the CRP: • Baseline developmental data for each of the species, e.g. thermal thresholds and day- degree requirements. • It is noted that there is a huge gap in knowledge on the application of this technique to species discrimination. • Unknown how this knowledge can be used on its own for species discrimination. - Progress made up to 3rd RCM in addressing identified gaps: • This area was extensively reviewed by A. Clarke (pers. comm.) and while potentially useful, is logistically challenging and was not pursued.

MALE LURE RESPONSE - Knowledge at the start of the CRP: • Each species nominated for this CRP respond to methyl eugenol (ME). • However some species are more sensitive to ME at low concentrations e.g B. dorsalis s.s. is more sensitive to low concentrations of ME than B. carambolae (Wee et al., 2002). • Initial feeding on high-concentration ME reduced response to subsequent exposure in B. dorsalis s.s . (Shelly, 1994). • The metabolites derived from being fed ME can be used to discriminate B. carambolae . - Gaps identified at the start of the CRP: • Lack of specific or detailed knowledge of the response of B. philippinensis and B. invadens . • The metabolites derived from flies being fed ME need to be tested in B. philippinensis. - Progress made up to 3rd RCM in addressing identified gaps: 42

• Male sex pheromone components (from rectal glands) of B. invadens and B. philippinensis following ME consumption have been identified and are the same as for B. dorsalis s.s. • Male sensitivity to ME in small cages complete and show that B. carambolae has much lower sensitivity relative to B. dorsalis, B. papayae, B. philippinensis and B. invadens . • The male mating advantage seen in the species following ME-feeding begins later for B. carambolae relative to B. dorsalis . • Lure sensitivity in field cage trials are underway.

PHEROMONE COMPONENTS AND RATIOS - Knowledge at the start of the CRP: • Pheromone components (both rectal gland and volatiles) following male consumption of methyl eugenol (ME) are exactly the same in laboratory cultures of each of three complex species: B. dorsalis s.s. and B. papayae. (Tan & Nishida, 1996; Wee & Tan, 2007). • Occurrence of wild hybrid flies (e.g. between B. papayae and B. carambolae ) in ME- baited traps. • Endogenous pheromone components are different between B. carambolae and both B. dorsalis and B. papayae. - Gaps identified at the start of the CRP: • Wild populations have not been tested to confirm the ratio of the pheromone systems between species. • Pheromone components of B. philippinensis and B. invadens have not been characterised. - Progress made up to 3rd RCM in addressing identified gaps: • Pheromone components post-ME feeding identified for laboratory-reared and wild males of B. invadens and B. philippinensis. These components are identical to those recorded for B. dorsalis and B. papayae . • Endogenous pheromone components for the above four species are different to those of B. carambolae. • Pheromone emissions analysed for B. dorsalis, B. papayae, B. philippinensis and B. carambolae reared under identical laboratory conditions. B. papayae and B. philippinensis profiles are virtually identical and B. carambolae emerges as clearly different. • The issue of pheromone ratios in wild flies has been discussed as a group. The consensus is that as differences in pheromone components are quantitative rather than qualitative between wild and lab flies, this issue will not be pursued as it is not considered critical to the question.

BEHAVIOUR - Knowledge at the start of the CRP: • Host preference and performance: knowledge for various individual Bactrocera populations/species from isolated studies. • Mating compatibility: knowledge for individual species crosses from isolated studies. Evidence for mating compatibility between a mix of wild/long-term cultures: B. papayae/B. carambolae ; B. dorsalis /B. invadens ; B. dorsalis /B. carambolae ; B. philippinensis /B. dorsalis .

- Gaps identified at the start of the CRP: • Host preference and performance: comparative knowledge across populations/species. • Mating compatibility: comparative knowledge across populations/species from across their geographic range acquired under equivalent/simultaneous conditions. - Progress made up to 3rd RCM in addressing identified gaps: • An assessment of published host lists has been made in a comparative sense among the species, but differences in sampling effort make such comparisons biologically difficult. The extreme logistic difficulties of doing this work in the field means that it has not been undertaken. • Comparative sexual compatibility studies have been completed in field cages at Seibersdorf for among ‘wild-ish’ (within F5) B. dorsalis s.s . (Thai origin) , B. papayae (Malaysian origin) , B. philippinensis (Philippine origin) , and B. carambolae (Suriname origin). Pre and post-zygotic (egg-hatch) data demonstrate complete compatibility among B. dorsalis s.s ., B. papayae, and B. philippinensis ; whereas all three of these species are relatively incompatible with B. carambolae. • Mating compatibility tests have also been undertaken between B. dorsalis s.s. (Thai origin) and B. invadens (Kenya origin) – results demonstrate compatibility between these two species, regardless of ME feeding. • B. dorsalis and B. carambolae remained relatively sexually incompatible following ME feeding by males. • Further compatibility tests (prezygotic and postzygotic to F2) have been completed between B. dorsalis from Wuhan (China) and Tandojam (Pakistan) and a new colony of B. invadens from Kenya. Full mating compatibility has been demonstrated between both B. dorsalis populations and B. invadens . Hybrid offspring obtained between both crosses are also viable to the F2 generation. • Extensive mating compatibility studies between populations of B. dorsalis s.s . (from 12 provinces; 6 regions) , B. papayae (6 provinces; 4 regions) and B. carambolae (5 provinces; 3 regions) conducted in Thailand. Current results completed for intra-specific intra-regional crosses. No evidence of incompatibility observed so far. • B. dorsalis and B. carambolae from the north are relatively sexually incompatible with conspecific populations from the south of Thailand. B. dorsalis from the north of Thailand is sexually compatible with B. papayae from the south of Thailand. B. papayae is compatible with B. carambolae in the north of Thailand, but not in the south of Thailand.

HOST RANGE - Knowledge at the start of the CRP: • Well documented for most pest populations/species being tested in this CRP, but not yet fully for B. philippinensis and B. invadens. • Host range data are constantly being updated in all regions. - Gaps identified at the start of the CRP: • Host range data not fully documented for B. philippinensis and B. invadens. - Progress made up to 3rd RCM in addressing identified gaps: • Host range assessment are continuously underway and new host records are being added for B. invadens in Africa. • It is recognised that the published host list for B. philippinensis is incomplete, but members of the CRP have no ability to add to the list.

DISTRIBUTION - Knowledge at the start of the CRP: • Distribution data provided in the original and revised species descriptions (Drew & Hancock, 1994). • Despite not explicitly defined as a ‘species character-state’ in original and revised descriptions, collection locality is considered in many operational keys to species. For example, B. dorsalis within Thailand is restricted to central/northern Thailand and B. papayae to southern Thailand, but new data from Seibersdorf suggests this is not correct. • Distributions may change over time. • New information obtained from this CRP may contribute to revised distribution lists. - Gaps identified at the start of the CRP: • Do the listed distributions provided in the species descriptions and revised descriptions remain valid? Particularly the hypothesised zone of transition between B. dorsalis s.s. and B. papayae on the Thai/Malay Peninsula? • Endemic range of B. invadens is not known. - Progress made up to 3rd RCM in addressing identified gaps: • There is no apparent zone of transition between B. dorsalis s.s. and B. papayae on the Thai/Malay Peninsula based on population genetic data (e.g. Fst values and demonstrated gene flow) and morphological data that supports that these are not different species, but rather a continuum. • Morphological and wing shape analysis suggests that the native range of B. invadens need not have been restricted to Sri Lanka but may have been more widely distributed across the Indian subcontinent. B. invadens has been officially documented in Bhutan. • CRP participants note that B. invadens has continued to spread throughout sub- Saharan Africa. • New specimens of B. philippinensis and B. papayae have been collected and examined from across the Indonesia Archipelago (R. A. I. Drew).

PROTEOMICS - Knowledge at the start of the CRP: • No proteomic descriptions available, nevertheless, proteomics may be a good approach to use for species discrimination, especially for proteins encoded by fast evolving genes. • Potentially useful if both proteomics and genomics are combined. • There are some proteins that may be species specific in Tephritidae, e.g. proteins in the male accessory gland. - Gaps identified at the start of the CRP: • No proteomic descriptions exist for B. dorsalis complex species. • No knowledge of this technique being used for species discrimination. • No potential species specific proteins identified for target species. - Progress made up to 3rd RCM in addressing identified gaps: • Pheromone binding proteins have been identified and characterised in C. capitata (as a model species) and for some of these proteins the specificity of the pheromone volatile component has been identified. • The transcriptome datasets are used to infer the proteomes of these species. While these can be stage specific, they are now used instead of classic proteomic approaches 45

for identifying classes of proteins critical for biological functions. Classic proteomics has not been and will not be further pursued to look for species specific markers.

METABOLOMICS - Knowledge at the start of the CRP: • None - Gaps identified at the start of the CRP: • Nothing has been done on Tephritids, but may have some potential for species discrimination. - Progress made up to 3rd RCM in addressing identified gaps: • Work is in progress in defining the genes and protein products involved in key metabolic pathways of B. dorsalis .

Specific Progress and Individual Research Plans

TAXONOMY AND MORPHOLOGY Qinge Ji - 5 year plan: • Establish colonies of populations of economic importance of the B. dorsalis complex from different regions for other work ( e.g. behavioural) with other members of the CRP where possible. • Confirm the species of B. dorsalis complex in mainland China based on morphological characters or with supporting evidence where available ( e.g. corresponding pheromone, molecular or behavioural information). - Plans for the first 18 months: • Survey the populations of B. dorsalis complex in different regions in mainland China. • Collect specimens of B. dorsalis complex by ME trapping and collecting damaged hosts. Specimens dry pinned for morphological screening with corresponding material alcohol preserved for future genetic screening. • Describe and illustrate the morphological variations if they occur in collaboration with R. A. I. Drew. - Plans for the second 18 months: • Continue to collect samples in different regions, covering all of mainland China. • Continue species descriptions with Prof. D. Drew. • Establish colonies of economically important B. dorsalis complex species for cross- mating studies. • Explore the potential to establish a culture of B. dorsalis s.s. at Seibersdorf (reared fresh from field). • Continue to maintain both dry pinned and alcohol collections. - Plans for the third 18 months: • Continue collection of fruit flies from locations of mainland China not sampled in previous years. • Continue the molecular analysis of B. dorsalis specimen collected. • Cross-mate the 8 laboratory strains established and assess egg hatch and other parametersof the offspring into three generations. - Progress made up to 3 rd RCM: • Samples collected from fruit and through the establishment of trapping networks with different traps types and lures from 13 provinces in China. • Over 35,000 specimens collected and sorted. 46

• Specimen identified and deposited at Fujian Agriculture and Forestry University. • Described new distribution records of known species and described some new species (some of which may fall in the B. dorsalis complex). • Seventeen species of Bactrocera (described and undescribed) were identified. • Five new Bactrocera species described. • Genetic structure of B. dorsalis flies was analysed, indicating that the mt COI gene within populations was greater than that between the different populations.

R.A.I Drew - 5 year plan: • Extensive morphometric analyses of aedeagus (and aculeus where available) measurements of five target species from specimens already housed in existing collections in Brisbane, in addition to material obtained through the CRP. This data will be correlated with geographic origin of samples. • Review existing host fruit database from fruit fly surveys across SE Asia. - Plans for the first 18 months: • Collaborate with Qinge Ji on morphological work from mainland China and prepare illustrations. • Provide diagnosis, under current taxonomic species definitions, on fly cultures established for behavioural experiments as undertaken by other members of the CRP: Seibersdorf, Thailand, and Malaysia research groups. N.b. minimum of 30 whole specimens of both sexes dry pinned required with 2-3 week turnaround on identification. - - Plans for the second 18 months: • Undertake a thorough morphological examination of B. dorsalis material received from China. This will include morphometric studies and also made available for molecular work where possible. • Provide diagnosis, under current taxonomic species definitions, on fly cultures established for behavioural experiments as undertaken by other members of the CRP. N.b. minimum of 30 whole specimens of both sexes dry pinned required with 2-3 week turnaround on identification. - Plans for the third 18 months: • Not defined. - Progress made up to 3 rd RCM: • Collaboration with China, Pakistan Sri Lanka, Indonesia and other locations in southeast Asia and the Sub-Indian continent on identification of samples. • Morphological revision undertaken based on good collections of B. invadens from Africa and compared these with original specimens of B. invadens from Sri Lanka; all samples examined to date are B. invadens . • Comprehensive taxonomic monograph on southeast Asian Bactrocera published. • Synonimization of B. philippinenesis with B. papayae .

Tony Clarke / Mark Schutze - 5 year plan: • Morphological and morphometric analyses of adults from all five target species in the Bactrocera dorsalis complex. • Morphological and morphometric analyses of juvenile stages (to be done by Gary Steck). - Plans for the first 18 months: 47

• Continued development of geometric morphometrics as a potential discriminatory and diagnostic tool. • Collection of material for dispersal to molecular workers in CRP. • Collaboration with R.A.I. Drew on morphometric data collation and analyses. - Plans for the second 18 months: • Finalise data analysis and publications. • Pending financial situation, address the B. tryoni complex through a multidisciplinary approach. • Construct specimen database for upload to DIR-SIT. - Plans for the third 18 months: • Finalise and publish work on B. invadens . • Contribute to the formal synonymization of B. invadens, B. papayae, and B. philippinensis with B. dorsalis. - Progress made up to 3 rd RCM: • Completed collections of material for B. dorsalis s.s., B. papayae, B. philippinensis and B. carambolae from appropriate locations in Taiwan, Thailand, Malaysia, Singapore and the Philippines. • Completed collection of material of B. invadens from five countries in Africa and the Indian Subcontinent. • Morphological and morphometric data analysis of B. invadens and B. dorsalis material collected from across their geographic range completed. • Geometric morphometrics using a generalised procrustes analysis shows a population structure that agrees with the single species hypothesis for B. dorsalis s.s., B. papayae, B. philippinensis , and that there is evidence of isolation by distance structured around the land-bridges of the South China Sea; B. carambolae is distinct. • All work on south east Asian target taxa of the B. dorsalis complex completed and published.

Sunday Ekesi / Fathiya M. Khamis - 5 year plan: • Collect and share biological material of B. invadens with other CRP participants as required. • Conduct morphometric analyses involving African, Sri Lankan, and Indian populations of B. invadens in relation to populations of the B. dorsalis complex. - Plans for the first 18 months: • Obtain collections of different populations of B. invadens from Africa, Sri Lanka and India for morphometric analysis and share with workers in CRP. • Conduct morphometric analyses involving African, Sri Lankan, and Indian populations of B. invadens . - Plans for the second 18 months: • Geometric morphometric and molecular markers to be generated for the collected species. - Plans for the third 18 months: • None. - Progress made up to 3 rd RCM: • B. invadens collected from Africa (Kenya, Uganda, Nigeria) and Sri Lanka. Other B. dorsalis complex species ( B. dorsalis s.s. and B. kandiensis ) collected from Hawaii and Sri Lanka. Outgroup species ( B. correcta,, B. cucurbitae, B. oleae and B. zonata ) collected from Sri Lanka, Kenya and Mauritius. 48

• Morphometric analysis completed, carried out in conjunction with COI gene confirmation.

BEHAVIOUR Tony Clarke/Mark Schutze/Seibersdorf staff - 5 year plan: • Specimens from each established culture confirmed by R.A.I. Drew. • Post-zygotic compatibility measured to egg-hatch of F1 from crosses. • Where possible, characterise additional elements of the mating system of B. tryoni complex as a model system for the target species ( e.g. acoustic communication, pheromone capture). - Plans for the first 18 months: • Carry out inter-specific cross-mating compatibility between at least 4 population/species ( B. papayae (Serdang*, Malaysia), B. dorsalis s.s. (Saraburi and Nakhon Si Thammarat, Thailand) , B. philippinensis (Guimaras Is., Philippines), and B. carambolae (Paramaribo, Suriname)). Collect data as per FAO/IAEA protocols ( e.g. compatibility indices). • Possible inclusion of Todd Shelly in carrying out remaining cross-mating trials at Seibersdorf. • Possible R&D for Todd Shelly on studies on ±ME effects in cross-mating trials. - Plans for the second 18 months: • Finalise data analysis and publications. • Complete studies on ME effects in cross-mating trials. • Pending financial situation, address the B. tryoni complex through a multidisciplinary approach. • Construct specimen database for upload to DIR-SIT. - Plans for the third 18 months: • Confirm B. kandiensis identity and complete compatibility study. • Seibersdorf maintaining colonies. - Progress made up to 3 rd RCM: • Inter-specific cross-mating compatibility and postzygotic studies between population/species of B. dorsalis, B. papayae, B. philippinensis, B. invadens, and B. carambolae complete. • Preliminary compatibility studies between B. kandiensis and B. dorsalis carried out at Seibersdorf. • ME male feeding did not reduce relative incompatibility between B. dorsalis and B. carambolae .

Suksom Chinvinijkul/Nidchaya et al. - 5 year plan: • Include B. carambolae as per other species. • Specimens from each established culture confirmed by R.A.I. Drew. • Post-zygotic compatibility measured from egg-hatch of F1 to pupation and sex-ratios from crosses. - Plans for the first 18 months: • Confirm that B. dorsalis s.s. is represented by one species in Thailand, by carrying out intra-specific cross-mating compatibility among six geographic populations of B. dorsalis s.s . Populations sourced from different hosts. 49

• Confirm that B. papayae is represented by one species in Thailand, by carrying out intra-specific cross-mating compatibility among three geographic populations of B. papayae . Populations sourced from different hosts. • Based on objectives 1+2, carry out inter-specific cross-mating compatibility tests between B. dorsalis s.s. and B. papayae . - Plans for the second 18 months: • Based on objectives 1 and 2, carry out inter-specific cross-mating compatibility tests between B. dorsalis s.s. and B. papayae . • Prioritise inter-specific cross-mating tests between geographically extreme populations (North-South, East-West) of B. dorsalis s.s., B. papayae (North-South) and B. carambolae. - Plans for the third 18 months: • Confirmation of identity of populations used in the Thai north-south mating compatibility studies using molecular markers and the completion of pheromone analysis. • Prepare publication for final CRP proceedings. - Progress made up to 3 rd RCM: • All B. dorsalis s.s. intra-region cross-mating complete. • All B. papayae and B. carambolae intra-region cross-mating complete. • All north and south inter-regional cross-mating studies complete. • B. dorsalis and B. carambolae from the north are relatively sexually incompatible with conspecific populations from the south of Thailand. B. dorsalis from the north of Thailand is sexually compatible with B. papayae from the south of Thailand. B. papayae is compatible with B. carambolae in the north of Thailand, but not in the south of Thailand.

A.K.W. Hee / S.L. Wee / K.H. Tan - 5 year plan: • Will focus on cryptic species response to ME (laboratory reared) and genetic analysis (in collaboration with Thai laboratory) of wild flies that have responded to ME. • Male cryptic species ( B. invadens , B. philippinensis , B. dorsalis s.s. and B. papayae ) behavioural response to ME. • Identification of putative antennal polypeptides specific to the above-mentioned species following exposure to ME, as means for possible use in species discrimination. • Original plan involving cross-mating studies has been abandoned due to serious biosecurity and ethical issues in conducting the outdoor field cage studies involving B. invadens , B. philippinensis and B. dorsalis s.s . • Molecular analysis outsourced to Thailand group. - Plans for the first 18 months: • Establishment of wild/wildish fruit fly culture of B. papayae and cultures of B. invadens , B. philippinensis and B. dorsalis s.s. (to be obtained from IAEA Seibersdorf). - Plans for the second 18 months: • Males of target species ( B. invadens , B. philippinensis , B. dorsalis s.s. and B. dorsalis s.l. ) assessed for behavioural response to ME using quantal response bioassays. • Identification of putative antennal polypeptides specific to the above-mentioned species following exposure to ME, as a proteomic technique for possible use in species discrimination. - Plans for the third 18 months: 50

• Maintain colonies for ongoing chemical ecology.

- Progress made up to 3 rd RCM: • Establishment of wild fly cultures of B. papayae and that of B. invadens , B. philippinensis and B. dorsalis s.s. successfully obtained from IAEA Seibersdorf under strict biosecurity containment. Flies used in chemical ecology (see below).

Sunday Ekesi / Fathiya M. Khamis - 5 year plan: • Investigate whether heritable life history differences exist between African and Asian populations of B. invadens. - Plans for the first 18 months: • Collection and establishment of colonies of African and Asian population. - Plans for the second 18 months: • Investigation of heritable life history differences among populations. - Plans for the third 18 months: • None. - Progress made up to 3 rd RCM: • Collections of B. invadens from Africa and a sample from Sri Lanka and other Bactrocera species including B. kandiensis (Sri Lanka), B. zonata (Mauritius), B. dorsalis s.s. (Hawaii), B. cucurbitae (Kenya), B. oleae (Kenya) and B. correcta (Sri Lanka). Samples from Sri Lanka of B. invadens and B. kandiensis were too few to be shared with members of the CRP.

CHEMICAL ECOLOGY Suksom Chinvinijkul et al. - 5 year plan: • Rectal-gland pheromone collection and analysis for B. dorsalis s.s. (from all six regions) and B. papayae (from all three regions) – from year 3 of CRP. Analysis outsourced to Malaysian group (Tan et al). - Plans for the first 18 months: Collection and forwarding of materials to Malaysia for rectal-gland pheromone collection and analysis. - Plans for the second 18 months: • Collection and forwarding of materials to Malaysia for rectal-gland pheromone collection and analysis. - Plans for the third 18 months: • Identification of the material provided for chemical ecology analysis to be confirmed by further independent evidence. Material from all crossing colonies to be sent to Mahidol University, and outstanding material to be sent to Alvin for remaining pheromone analysis. - Progress made up to 3 rd RCM: • Material has been collected and sent to Malaysia for rectal-gland pheromone collection and analysis.

S.L. Wee / A.K.W. Hee / Ritsuo Nishida / K.H. Tan - 5 year plan: • Rectal gland analysis on material collected and supplied by Thai group (see above). 51

• Pheromone collection (pheromone gland (+/-ME fed) and headspace) and analysis for wild/wildish individuals of all five target species.

- Plans for the first 18 months: • Pheromone collection (pheromone gland (+/-ME fed) and headspace) and analysis for wild/wildish individuals of B. carambolae and B. papayae. • Components and ratios measured. - Plans for the second 18 months: • Pheromone collection (pheromone gland (+/-ME fed) and headspace) and analysis for wild/wildish individuals of the five target species. • Determination of the pheromone system of the field morphohybrids. - Progress made up to 3 rd RCM: • Sex pheromones of B. invadens and B. philippinensis following ME consumption have been identified. • Headspace sampling for B. invadens, B. philippinensis, B. dorsalis and B. papayae male pheromone emissions with and without ME consumption has been analysed. Quantitative and qualitative analysis of ME related pheromone components has been completed. The ratio of the two components ( E)-coniferyl alcohol and 2-allyl-4,5- dimethoxy phenol were found to be identical across the four species. • Analysis of material supplied by Thai collaborators showed inconsistency with morphological identifications of species used in mating compatibility studies in Thailand. - Plans for the third 18 months: • Complete headspace analysis of male pheromone emissions of ME fed and unfed flies. • Following the confirmation of identifications of Thai material, resolve inconsistency previously detected in Thailand. • Complete the probit bioassay of males of the four species to ME to measure relative dose response. • Complete analysis of the ME exposed male antenna protein profiles of the four species.

P. Teal/I ul Haq/Seibersdorf - Plans for the third 18 months: - Complete work on the sensitivity of B. dorsalis, B. carambolae and B. invadens to different doses of ME in field cages. - Confirm qualitative and quantitative profiles of male released volatiles of B. invadens, B. philippinensis, B. papayae, B. dorsalis and B. carambolae to assess species limits within the target taxa. - Analyse male pheromone emissions collected from male flies exposed to different amounts of ME. - Prepare flies for male antennal protein extracts. - Progress made up to 3 rd RCM: - Initial work on the sensitivity of B. dorsalis, B. carambolae and B. invadens to different doses of ME has been completed in field cages. - Initial qualitative and quantitative profiles of male released volatiles of B. philippinensis, B. papayae, B. dorsalis and B. carambolae to assess species limits within the target taxa.

MOLECULAR AND GENETIC 52

Karen Armstrong/QUT - 5 year plan: • Undertake phylogenetic and other genetic analyses of the sequence data. • Based on the inferred species delineations from molecular and behavioural data, retrospective identifications will be sought from R.A.I. Drew. • Based on the inferred species delineations from molecular and behavioural data, molecular diagnostic markers will be developed. - Plans for the first 18 months: • Screening mtDNA and nDNA genes for geographic populations of at least the following four species: B. dorsalis s.s. (Thailand, Taiwan), B. papayae (Malaysia, Indonesia) B. carambolae (Suriname, Malaysia, Indonesia) and B. philippinensis (Philippines). Specimens provided by TC/MS through Australia. • Collaboration with Norman Barr in genetic studies. - Plans for the second 18 months: • Finalise data analysis and publications. • Pending financial situation, address the B. tryoni complex through a multidisciplinary approach. • PCR population analysis of selected SNP regions identified through comparative transcriptome analysis. - Plans for the third 18 months: • Publish comparative transcriptome dataset and related SNP and phylogenetic analysis. • Provide transcriptome dataset from several species/populations to Anna Malacrida to identify the specific genes related to reproduction and behaviour. • Confirmation of identity of B. carambolae, B. papayae and B. dorsalis populations used in Thai north-south mating compatibility studies using molecular markers. - Progress made up to 3 rd RCM: • All four target taxa plus B. invadens and outgroups ( B. occipitalis, B. cacuminata, B. opiliae, B. kandiensis, B. musae, B. tryoni ) have been screened for two mtDNA and two nDNA and two rDNA regions. • Data has been incorporated into a single-gene and combined-gene phylogenetic analysis. The COI and ITS1 gene regions show B. carambolae and B. occipitalis as distinct relative to the other four species. • Collaboration with Norman Barr on the barcoding project is being maintained. • Comparative transcriptome data for B. dorsalis s.s. , B. papayae, B. philippinensis, B. carambolae and B. invadens have been analysed for ‘species’ specific SNPs. • Using wild populations, new species specific SNP markers have been found for B. carambolae and B. occipitalis . • Phylogenetic analysis using loci with polymorphic SNPs has been carried out for B. dorsalis s.s. , B. papayae, B. philippinensis and B. carambolae.

Anna Malacrida - 5 year plan: • Microsatellite analysis of B. dorsalis s.s. (Thailand, China, and other SE Asian locations) and B. invadens (Sub-Saharan Africa and Sri Lanka). • For at least one species, potential EST library development for specific relevant tissues associated with behavioural or reproductive biology (from year 3). - Plans for the first 18 months: • Extension of microsatellite analysis to specimens provided by Karen Armstrong and Nidchaya Aketarawong. 53

- Plans for the second 18 months: • Publish the work on B. dorsalis s.s. • Screen other samples from China if they become available. • Derive specific marker for B. dorsalis s.s. from y chromosomes. • Using the C. capitata transcriptomes, odor and pheromone binding genes that have been identified, compare B. dorsalis s.s. transcriptomes to identify homologous genes . - Plans for the third 18 months: Complete molecular and functional characterisation of the Y-chromosome of B. dorsalis s.s. (Saraburi) and to extend this characterisation to wild populations and to other target taxa of the B. dorsalis complex. This will be done in collaboration with Mahidol University (Thailand). • Undertake bioinformatics analysis of the transcriptome dataset from several species/populations provided by Karen Armstrong to identify the specific genes related to reproduction and behaviour. • Provide sequences to Antigone Zacharopoulou to be used for in situ hybridisation for chromosome mapping. - Progress made up to 3 rd RCM: • The genotyping of six Chinese populations has been performed for B. dorsalis s.s. • Database of SSR fingerprint of 20 populations of B. dorsalis s.s. across the species range has been constructed. This allows us to potentially identify the origin of new outbreaks of B. dorsalis s.s. • Specific Y-chromosome sequences have been identified in B. dorsalis s.s. (Saraburi lab strain, Seibersdorf). SNPs within one of them have been identified. Transcribed sequences have been identified.

Antigone Zacharopoulou / Antonios Augustinos / Elena Drosopoulou / Kostas Bourtzis - 5 year plan: • Polytene chromosome analysis of the five target species (as listed previously) from colonies in Seibersdorf. • Same for F1 progeny or subsequent generations if available. • Upon variability of the strains assess the need to analyse more fly strains at the chromosome level (beyond 18 months). • Depending on the chromosomal maps obtained, diagnostic characters for each species will be developed accordingly. • Given the opportunity, genomic information developed elsewhere (from within or outside from the CRP) will be mapped onto the chromosomes. - Plans for the first 18 months: • Screening using mitotic polytene chromosomes from established colonies. • Chromosome distribution of the two elements: piggybac and Hopper elements in the same materials from point 1 through in situ hybridisation. - Plans for the second 18 months: • Analysis of polytene chromosomes of more F1 hybrids depending on availability of material at Seibersdorf. • As a priority and where possible, in situ hybridisation of uniquely localised markers for the five target members of the complex, for example genome project derived data, ESTs – in collaboration with genomics groups. • Possibly more extended in situ hybridisation of piggyBac and Hopper for more samples from more populations depending on availability of material at Seibersdorf. 54

• Polytene chromosome analysis of closely related outgroup species, depending on availability of material at Seibersdorf. - Plans for the third 18 months: • Publish the results from comparative chromosome analysis. • Enrich cytogenetic maps using new sequences including those provided by Anna Malacrida. • Analyse and compare the complete mitochondrial genomes of the five target taxa of the B. dorsalis complex. • Include outgroup ( B. kandiensis or B. occipitalis ) from target taxa for comparison of chromosome analysis. - Progress made up to 3 rd RCM: • Comparison of B. papayae, B. philippinensis, B. invadens and B. carambolae to the reference map of B. dorsalis s.s. has been done, showing no striking differences. • Polytene chromosome analysis of bidirectional F1 hybrids of B. dorsalis s.s. and B. invadens, and B. dorsalis s.s. and B. carambolae has been completed and no detectable differences have been found except for few minor asynapses in the hybrids of B. dorsalis and B. carambolae . • Preliminary in-situ hybridisation of piggyBac and Hopper in the five target members of the complex has been performed. Six unique genes have been mapped on the polytene chromosomes of B. dorsalis and the above hybrids.

Nidchaya Aketarawong et al. - 5 year plan: • Complete the genetic analysis of B. dorsalis species complex and examine congruence with other types of evidence and diagnostic characters. - Plans for the first 18 months: • Complete screening, start applying microsatellite DNA typing and carry out genetic analysis of B. dorsalis species population samples from the following theme: o B. dorsalis species complex colonies from Seibersdorf. o Wild B. dorsalis species complex population samples across geographical range. o Wild B. dorsalis species complex population samples from Malaysian Peninsula. • Begin providing genotyping and genetic analyses for the other group sample: o Behaviour: samples from Suksom et al . o Chemical ecology: samples from Suk Ling Wee. o Taxonomy. • Increase potential to develop and evaluate novel molecular markers, diagnostic tools. - Plans for the second 18 months: • Collect B. carambolae from Thai/Malay Peninsula and Indo/Malay Archipelago. • Collect B. carambolae from collaborators. • Develop and evaluate molecular markers for B. carambolae populations. - Plans for the third 18 months: • Characterize Y-chromosome in different populations/species of B. dorsalis s.s. and B. carambolae • Confirmation of identity of populations used in north-south mating compatibility studies using molecular markers. • Try to collect samples of B. carambolae from wider georgraphic distribution and subject to genetic tests. 55

- Progress made up to 3 rd RCM: • Completed collection and genotyping of B. dorsalis s.s. and B. papayae populations across southern Thailand and the Malay Peninsula using microsatellite markers. • Analysis of population genetics based on these microsatellites inferred significant gene flow between sympatric and allopatric populations. Also isolation-by-distance was not observed across southern Thailand and the Malay Peninsula. • Microsatellite DNA markers tested on B. dorsalis s.s. and B. papayae didn’t show fixed alleles consistent with taxa-specific identification. • Four specific Y-chromosome sequences (contig 1, 2, 3, and 4) have been identified in B. dorsalis s.s. (Saraburi lab strain, Seibersdorf). These sequences have been tested in four populations of B. dorsalis s.s. (three lab strains and one wild). No differences were found for male specific amplification. • Two populations of B. carambolae from Thailand (north and south), one from Malaysia and three from Indonesia (Java, Sumatra, Borneo) were received. Of these, only one sample from Thailand (south) and one from Sumatra were tested for male specificity of these Y-chromosome sequences. In the wild sample from Indonesia, only contig 2 display a male specific pattern. In the sample from Thailand, the amplification patterns indicate no evidence of male specificity for the four contigs. These preliminary data seem to indicate the differences between the Thailand and Indonesia samples.

Sunday Ekesi / Fathiya M. Khamis - 5 year plan: • Obtain samples of B. invadens populations from Africa, and Sri Lanka including colonies already established and contribute to the development of a population genotype database (in collaboration with A. Malacrida). • Evaluate DNA variation using mitochondrial and nuclear gene sequences and compare populations for evidence of population structure and potentially distinct gene pools. • Collect and share biological material of B. invadens with other CRP participants as required. - Plans for the first 18 months: • Obtain collections of different populations of B. invadens from Africa and Sri Lanka for DNA variation analysis, population genotype database and sharing with workers in CRP. • Screen different populations for one mitochondrial (COI) and nuclear (period) gene. - Plans for the second 18 months: • Other molecular markers will be used for confirmation ( e.g. period gene). - Plans for the third 18 months: • None. - Progress made up to 3 rd RCM: • Collections of B. invadens from Africa and a sample from Sri Lanka and other Bactrocera species including B. kandiensis (Sri Lanka), B. zonata (Mauritius), B. dorsalis s.s. (Hawaii), B. cucurbitae (Kenya), B. oleae (Kenya) and B. correcta (Sri Lanka). • The morphometric analyses were confirmed with COI gene.

Ceratitis FAR Complex

Background Situation Analysis

The Afro-tropical group of fruit flies, Ceratitis fasciventris, C. anonae and C. rosa (FAR) , together with C. capitata , are considered to be among the major horticultural pests of that region (White & Elson-Harris 1992; De Meyer 200la) and these species are of quarantine significance (EPPO/CABI 1997). They are highly polyphagous and damage a wide range of unrelated wild and cultivated crops (De Meyer et al. 2002a), resulting in enormous economic losses wherever they occur (Barnes 2000; Lux 2000; De Meyer 2001b).

Ceratitis rosa, C. fasciventris and C. anonae are considered the three members of the Ceratitis FAR species complex (Virgilio et al . 2007a, 2007b). From the taxonomic point of view, C. fasciventris was considered to be a variety of C. rosa (Bezzi 1920). Only recently has it been recognized as a different entity with species status (De Meyer 2001a).

Unlike C. capitata , which has spread from its home range in East Africa and attained an almost world-wide distribution over the last century (Fletcher 1989; White & Elson-Harris 1992), C. rosa, C. fasciventris and C. anonae have so far not been reported outside the African continent (except La Réunion and Mauritius) but are potentially invasive.

Due to the difficulty in distinguishing some members of the complex morphologically, a number of molecular approaches for species recognition were used (Baliraine et al ., 2004; Barr et al ., 2006; Virgilio et al ., 2008). However these remain inadequate for quarantine purposes and much more robust molecular markers are needed.

Baseline and Progress on the Ceratitis FAR Complex

DNA - Knowledge at the start of the CRP: • Attempts to develop specific diagnostic markers were made but they are so far ineffective. - Gaps identified: • There is the need for further exploration for markers, especially regarding microsatellites. - Progress up to 3 rd RCM in addressing identified gaps: • Microsatellites were developed for the Ceratitis FAR complex. African populations for all three species were analysed. Population genetic structure for the complex revealed five clearly distinguishable clusters: C. fasciventris : F1, F2; C. anonae : A; C. rosa : R1, R2. • There is still the need for a diagnostic marker, either as a sequence (explore ITS2) or banding pattern (microsatellites).

CYTOLOGY - Knowledge at the start of the CRP: • No data available. Cytology has the potential to provide a diagnostic tool. - Gaps identified: • Need for researcher to conduct cytological studies of this complex.

- Progress up to 3 rd RCM in addressing identified gaps: 58

• No studies conducted so far.

ALLOZYMES - Knowledge at the start of the CRP: • Previous work conducted by Malacrida et al . (1996) only on C. rosa (perhaps also including C. fasciventris ). - Gaps identified: • No further work considered under the CRP; covered by DNA work.

GENOMICS - Knowledge at the start of the CRP: • Current focus on C. capitata. Y chromosome sequences are available. Transcriptomics from different sexes and different stages are available. This allows identifying genes related to odorant binding proteins, pheromone binding proteins and genes correlated to spermatogenesis and male accessory glands. - Gaps identified: • This knowledge is not available for representatives of the Ceratitis FAR complex; such study can be considered. - Progress up to 3 rd RCM in addressing identified gaps: • No further studies conducted.

MORPHOLOGY - Knowledge at the start of the CRP: • Males can be distinguished to some extent. - Gaps identified: • Separation of females is difficult. • Larval morphology not studied. - Progress up to 3 rd RCM in addressing identified gaps: • After recognition of five clusters (based on molecular work), adult characters for males (and to lesser extent for females) were re-examined. Consistent morphological differences were found to distinguish male C. rosa (R1-R2) and C. fasciventris (F1- F2). Male morphological differences were used to re-examine and assign available C. rosa and C. fasciventris specimens to the different genotypes. • Larval morphology for representatives of each taxonomically recognized species within the Ceratitis FAR complex was studied. SEM studies for all immature stages were conducted. Within C. rosa unusual larval findings contradict earlier observations based on South African populations. No distinct differences are observed between R1 and R2 but more detailed investigation of larval morphology is required.

MORPHOMETRICS - Knowledge at the start of the CRP: • No data available. - Gaps identified: • Morphometric study on females might have potential for separation of females. - Progress up to 3 rd RCM in addressing identified gaps: • Morphometric study was conducted on all five genotypes, using wing landmarking and wing banding. Wing landmarking provides some separation between the three FAR species and, to a lesser extent, the five genotypes for both sexes. Wing banding is providing no clear resolution. 59

MALE LURE RESPONSE - Knowledge at the start of the CRP: • All males are attracted to trimedlure. - Gaps identified: • Response of representatives of the Ceratitis FAR complex to male lures to be investigated. - Progress up to 3 rd RCM in addressing identified gaps: • Response of representatives of the Ceratitis FAR complex to EGOlure was investigated in Tanzania and La Réunion. EGOlure has been shown to be a stronger attractant for C. rosa (R1 and R2) than trimedlure in Tanzania. Data of the Réunion study is under analysis.

DEVELOPMENTAL PHYSIOLOGY - Knowledge at the start of the CRP: • Developmental physiology has not been used for diagnostic purposes. - Gaps identified: • Given the recent progress in the genetic structure of the complex, there is the need for additional studies to determine whether there are developmental/ physiological differences between the entities recognized. - Progress up to 3 rd RCM in addressing identified gaps: • These studies have started for the two C. rosa types. There is a positive linear relationship between temperature and development rate. A different upper temperature threshold was observed but not a lower, with the R1 type being more tolerant to higher temperature.

BEHAVIOUR - Knowledge at the start of the CRP: • Some behavioural work on C. rosa , limited for the other entities. • Limited experiments under forced laboratory conditions have shown that all taxonomically recognized species of the FAR complex can interbreed (providing viable offspring for cross-mating between C. rosa and C. fasciventris ). - Gaps identified: • Mating compatibility studies under semi-natural field cage conditions can contribute to resolving the specific status of the taxa within the complex. • Given the recent progress in the genetic structure of the complex, there is the need for additional studies to determine whether there are behavioural differences between the entities recognized. - Progress up to 3 rd RCM in addressing identified gaps: • Mating compatibility studies have started for the two C. rosa entities and appear to show some reproductive isolation between the two.

PHEROMONE COMPONENTS - Knowledge at the start of the CRP: • Pheromones of entities recognized not studied.

- Gaps identified: 60

• Pheromone component studies can contribute to resolving the specific status of the taxa within the complex. • Given the recent progress in the genetic structure of the complex, there is the need for additional studies to determine whether there are pheromone differences between the entities recognized. - Progress up to 3 rd RCM in addressing identified gaps • Pheromones were investigated and preliminary identified for all three FAR species. All species share some components but also demonstrate specific elements. • The sex pheromones of both species are multicomponent. The composition of investigated pheromones is different from that of C. capitata , except for some components like farnesene.

HOST RANGE - Knowledge at the start of the CRP: • Host range is well known for representatives of the complex. - Gaps identified: • Given the recent progress in the genetic structure of the complex, there is the need for additional studies to determine whether there are host range differences between the entities recognized. - Progress up to 3 rd RCM in addressing identified gaps: • Host range for the two F and two R entities have been re-examined based upon existing records. Information for the F entities is inconclusive because of lack of data for F1. R1 and R2 do not show major differences in host range except that the host from temperate climates ( Pyrus , Rubus , Coffea ) are predominantly infested by R2.

DISTRIBUTION - Knowledge at the start of the CRP: • Distribution patterns are well known for representatives of the complex. - Gaps identified: • Given the recent progress in the genetic structure of the complex, there is the need for additional studies to determine whether there are distributional differences between the entities recognized. - Progress up to 3 rd RCM in addressing identified gaps: Distribution range has been re-examined based upon existing records. R1 distribution is limited by latitude (absent southern part of the African continent) and altitude (absent from higher altitudes). F1-F2 largely geographically separated (western-central versus eastern Africa). However, isolated populations of ‘western’ type found in Angola, Malawi, Tanzania, Zambia. New altitudinal transect study has been started in Tanzania to reveal seasonal changes and altitudinal limitations.

CUTICULAR HYDROCARBONS - Knowledge at the start of the CRP: • Cuticular hydrocarbons of the Ceratitis FAR complex not studied. - Gaps identified: • Cuticular hydrocarbon studies can contribute to resolving the specific status of the taxa within the complex. • Given the recent progress in the genetic structure of the complex, there is the need for cuticular hydrocarbon comparisons to determine whether there are differences between the entities recognized. 61

- Progress up to 3 rd RCM in addressing identified gaps: • Cuticular hydrocarbons of both sexes (virgin, 20 days old) were analyzed. Specific differences were detected between the three taxonomically recognized species of the complexes. Sexual differences were also found in each species. The CHC from the two C. rosa (R1, R2) – from Kenya, are obtained and the CHC of males and females of C. rosa R1 will be analysed by the means of GCxGCMS during September and October 2013.

Specific Progress and Individual Research Plans: Ceratitis FAR Complex General observation: Given the difficulties in establishing a colony of C. fasciventris F1 type, it is deemed not feasible to gather all information from the different approaches to differentiate between F1 and F2. Therefore, the focus is on R1-R2 differentiation, and further F1-F2 differentiation is not considered. Should a F1 colony be forthcoming in the near future of the last 18 months, inclusion of F1-F2 will be re-considered.

TAXONOMY Marc de Meyer - 5 year plan: • Provide diagnostic markers to differentiate adults of both sexes for species within the complex (except below). • In collaboration with Fellow from Seibersdorf laboratory: provide morphometric diagnostic markers to differentiate females within the complex - Plans for the last 18 months: • Explore the possibility of using the most significant differences in wing landmarking, as defined by the multivariate analysis, into a suitable identification tool to differentiate the females of the three FAR species and five genotypes. • Continue re-examination of taxonomic collections for further assignment of collection specimens to the different entities. - Progress made up to 3 rd RCM: • After recognition of five clusters (based on molecular work), adult characters for males (and to lesser extent for females) were re-examined. Consistent morphological differences were found to distinguish male R1-R2 and F1-F2. Male specimen holdings in taxonomic collections (Royal Museum for Central Africa Tervuren, Natural History Museum London, Stellenbosch University Stellenbosch, South African Collections Pretoria) were re-examined and assigned to one of the five genotypes. • Morphometric study, based on wing landmarking and wing banding was conducted by a MSc student of the University of Antwerp (J. Van Cann) for specimens used in genotypic study. Results show clear sexual dimorphism in all five genotypes. Wing landmarking allow separation to a certain extent of all three FAR species and, less so, of the five genotypes.

Sunday Ekesi / Fathiya M. Khamis - 5 year plan: • Collect and share biological material of the FAR group with other CRP participants as required . - Plans for the last 18 months: • Establishment of R2 in Tanzania and further sharing of material with participants as required (pheromones, endosymbiosis) will be done. • Attempts will be made to establish a C. fasciventris colony from western Africa. • Material of South African collections will also be shared with partners on larval morphology to clarify earlier observed differences. - Progress made up to 3 rd RCM: • Colonies for the three taxonomically recognized species were established at ICIPE (Kenya) and the material was shared with other teams for pheromones/hydrocarbons/larval taxonomy/DNA research. 63

• Furthermore colonies for R1-R2 were established at ICIPE (Kenya): lowland population of Ceratitis rosa from guava fruits collected from Mwanjamba, Msambweni, Kenya and the highland populations was reared from mango fruits collected from Kithoka, Meru, Kenya and material shared with partners working on larval morphology and cuticular hydrocarbons. • Colony of R2 from Tanzania at SUA is progressing well but difficulties are encountered with the colony from R1 and we have resorted to use environmental chamber because of the low ambient temperatures. • A third partner (Citrus Research International, South Africa) is also in the process of establishing colonies of both R1 and R2 from South Africa to conduct studies on developmental physiology and mating compatibility in conjunction with the Kenyan and Tanzanian partners.

Gary Steck / Sunday Ekesi - 5 year plan: • To provide indication whether morphological characters of immature stages (larvae) provide diagnostic character for differentiation. - Plans for the last 18 months: • Conduct an in depth study of larval morphology for all entities currently available. Supplement the R1-R2 with material from South Africa to resolve the issue of contradictory information of an earlier study. This study will be conducted by a PhD student from ICIPE in collaboration with Gary Steck. - Progress made up to 3 rd RCM: • Larval morphology for representatives of each taxonomically recognized species within the FAR complex was studied. • SEM studies for all immature stages were conducted. • Within C. rosa unusual findings contradict earlier observations based on South African populations. • A sample of R1 3 rd instar larvae, originating from the coastal region of Kenya was received and examined. Preliminary SEM examination shows them to be similar to the Kenyan highland population, i.e., oral ridge accessory plates are present also in the coastal population.

BEHAVIOUR Maulid Mwatawala / Sunday Ekesi - 5 year plan: • If necessary: to provide additional information on the specific status in case of conflicting data from other work packages using mating compatibility tests. - Plans for the last 18 months: • Mating compatibility, both pre and post zygotic will be further conducted in Kenya and Tanzania (pending establishment of the colonies). Contacts will be made with CRI (South Africa to enquire whether they will also conduct these tests). • Behavioural studies (including mating, responses to attractant etc) with regard to the highland population of C. rosa collected from coffee (Ruiru) has previously been documented (Pontiano Nemeye’s thesis) but no information is available with regard to the lowland population. • In addition to assessing mating compatibility, observation on courtship repertoire/mating behaviour will be recorded in field cages. Similarly, responses of the 64

two populations to trimed lure, EGOlure and synthetic food attractants will be documented in field cages. - Progress made up to 3 rd RCM: • Protocols on mating compatibility tests have been exchanged between Kenya and Tanzania. Studies have started in Kenya: Only 3 replications have been conducted and the results obtained so far showed some degree of mating incompatibility between the 2 populations of C. rosa and also between the 2 populations and C. fasciventris . The ISI values ranged from 0.75 to 0.90 with the Highland C. rosa x the Lowland C. rosa showing the highest degree of isolation. More replications are planned for September when the colony population increases. Tanzania: The cages for this study have been designed and the trials will start as soon as fairly large colonies of the two C. rosa type have been established.

CHEMICAL ECOLOGY Blanka Kalinova/ Lucie Vanickova - 5 year plan: • To improve the efficiency of new and sustainable control methods by means of use the sex pheromone and cuticular hydrocarbon profile of Ceratitis spp. of the FAR complex. - Plans for the last 18 months: • Pheromone analysis: obtain material of R1 and R2 at beginning of 2014 to study sex pheromone patterns. Obtain additional material of F2 and A at mid-2014 to complete the EAG analysis. - Progress made up to 3 rd RCM: • Pheromones were investigated and preliminary identified for C. rosa and C. anonae . The sex pheromones of the species are multicomponent. Both species share some components but also demonstrate specific elements. The composition of investigated pheromones is different from that of C. capitata , except for some components like farnesene. • Cuticular hydrocarbons of both sexes (virgin, 20 days old) were analysed. Specific differences were detected between the three taxonomically recognized species of the complexes. Sexual differences were also found in each species. The CHC from the two C. rosa (R1, R2) – from Kenya, are obtained and the CHC of males and females of C. rosa R1 will be analysed by the means of GCxGCMS during September and October 2013 and the results will be compared with the results of chemical analyses of C. rosa R2.

MOLECULAR Hélène Delatte / Massimiliano Virgilio / David Haymer / Anna Malacrida - 5 year plan: • Provide discriminant markers that can be used as a diagnostic tool to differentiate the three species of the complex. - Plans for the last 18 months: • David: Exploration of utility of ITS2 as a diagnostic markers using about 50 samples used for the genotyping (5 individuals x 2 populations x 5 genetic entities). Send material end of 2013, beginning of 2014. • Anna: use sequences from C. capitata to derive variable changes to the others for the FAR complex. 65

• Hélène/Massimiliano: To evaluate the use of microsatellites as a diagnostic tool by looking at specific banding patterns. • Hélène: To look for endosymbionts (i.e. Wolbachia ) in R1 and R2 populations (specimens from established colonies and specimens used for genotyping) to see whether any differences in presence/absence/strains are observed. Repeat this study for F1, F2 and A populations. - Progress made up to 3 rd RCM: • Microsatellites were developed for the FAR complex. • African populations for all three species were analysed. • Population genetic structure for the complex revealed five clearly distinguishable clusters (F1, F2, A, R1, R2). These results were published.

DEVELOPMENTAL PHYSIOLOGY Maulid Mwatawala / Sunday Ekesi - 5 year plan: • Compare developmental/physiological differences between the five entities recognized, with emphasis on R1-R2 differentiation. - Plans for the last 18 months: • Continue studies on Kenyan populations. • Start studies on Tanzanian and South African populations. - Progress made up to 3 rd RCM: • Preliminary studies conducted on R1-R2 differentiation with Kenya populations. Preliminary studies on thermo-tolerance for the two populations of C. rosa have been conducted. The linear regression model showed a strong positive linear relationship between temperature and development rate for all stages. Lower development threshold for all stages were estimated. Parameter estimates for the Brière-1 nonlinear model predicted the lower and upper temperature thresholds for all stages. This experiment will need to be repeated. • Exchange of protocols between Kenya, Tanzania, South Africa (Ekesi, Manrakhan, Mwatawala) is in progress.

HOST RANGE Marc De Meyer / Mwatawala - 5 year plan: • Redefine host records with regard to the five recognized entities within the FAR complex. - Plans for the last 18 months: • Carry out a further review of existing host records. No focused research on new host records is planned. - Progress made up to 3 rd RCM: • Re-evaluation of known host records based upon specimens study of natural history collections. Inconclusive results as all genotypes appear to be polyphagous. Tendency for R2 to be recorded from some temperate climate fruits for which there are no records for R1 (linked to distribution patterns, Cf below)

DISTRIBUTION Marc De Meyer/Maulid Mwatawala - 5 year plan: 66

• Redefine distribution records with regard to the five recognized entities within the Ceratitis FAR complex. - Plans for the last 18 months: • Carry out a further review of existing geographical records. • Continue sampling along altitudinal transect and analysis of data. - Progress made up to 3 rd RCM: • Re-evaluation was carried out of known distribution records. Available specimens were re-examined and divided according to the 5 groupings. • R1 distribution is limited by latitude (absent southern part of the African continent) and altitude (absent from higher altitudes). • F1-F2 largely geographically separated (western-central versus eastern Africa). However, isolated populations of ‘western’ type found in Angola, Malawi, Tanzania, Zambia. • New altitudinal transect study Tanzania started to reveal seasonal changes and altitudinal limitations. This study has been conducted for three months along an altitudinal transect, at 10 locations, about 100 m apart (altitudinal). Material is currently sorted, in order to determine the overlap/ cut-off points.

E. Logical Framework

Table 3.

Logical Framework

NARRATIVE OBJECTIVE MEANS OF IMPORTANT SUMMARY VERIFIABLE VERIFICATION ASSUMPTIONS INDICATORS

OVERALL OBJECTIVE

To assist Member Fruit and vegetable States in achieving production in Member sustainable fruit and States continue to suffer vegetable production N/A N/A major losses due to and in overcoming endemic and introduced constraints to SIT fruit flies of economic application and importance. international trade through the International trade in resolution of cryptic fruit and vegetable species complexes of commodities continue to major tephritid pests. increase and be disrupted by fruit fly pests, requiring expensive pre- and post-harvest treatments and quarantine measures.

International trade continues to suffer from unnecessary restrictions due to confusion about the biological identity, geographic distribution and lack of diagnostic tools for pest fruit fly species.

In contrast to pesticides, a more environmentally friendly pest fruit fly control requires precise applications based on the knowledge of the biology of species. 68

The area-wide integrated management of fruit flies of economic importance, including where appropriate SIT as the non-polluting suppression/eradication component, will increasingly be a cost- effective method to deal with fruit flies of economic importance.

SPECIFIC RESEARCH OBJECTIVE

Identify and define Basic and applied Sampling protocols, There are multiple the biological limits research that diagnostic tools, species present within of closely related defines biological species descriptions the single A. fraterculus pest species within species limits of and geographic nominal species. This the South American species within the distributions, constrains trade and SIT fruit fly ( A. A. fraterculus trapping and application. fraterculus ) complex. complex and ecological Facilitate the develops tools to information, taxonomic revision distinguish them presented in reports and develop so as to facilitate and publications. corresponding trade and SIT diagnostic tools to implementation. distinguish these species. Identify and redefine Basic and applied Sampling protocols, There is no universal the biological limits research that diagnostic tools, agreement on the validity and species status of defines biological species descriptions of the currently described five closely related species limits and geographic pest species within the B. species of economic within the B. distributions, dorsalis complex. This importance within dorsalis complex trapping and constrains trade and SIT the Oriental fruit fly and develops tools ecological application. (B. dorsalis ) to distinguish them information, complex. Develop so as to facilitate presented in reports corresponding trade and SIT and publications. diagnostic tools to implementation. distinguish these species. Determine the Mating Sampling protocols, There are concerns that variability within the compatibility, diagnostic tools, there may be intra- melon fly ( B. morphological and geographic specific differences cucurbitae ) regarding genetic variability distributions and which would interfere geographic and/or assessed so as to ecological with efficient SIT host races among allow efficient information, applications and trade. 69 populations from application of SIT presented in reports Africa, Indian Ocean to B. cucurbitae in and publications. and Hawaii. these regions. Identify and redefine Basic and applied Sampling protocols, Recent evidence the biological limits research that diagnostic tools, indicates the existence of and species status of defines biological species descriptions different entities within the members of the species limits and geographic the recognized species of Ceratitis FAR within the distributions, the Ceratitis FAR complex. Ceratitis FAR trapping and complex. Existing Develop more robust complex and ecological diagnostic tools for these species markers and develops tools to information, entities are not suitably identification tools distinguish them presented in reports robust for quarantine use. for the members of so as to facilitate and publications. the complex. trade and Diagnostic tools, protection of reports and agriculture through publications. improved quarantine services.

OUTPUTS A. fraterculus

1. Establish a Protocols Protocol available Quality of outcomes network for developed and to all investigators. highly dependent on targeted, network essential co-ordination structured established for between groups working sampling of the structured on A. fraterculus . complex together collecting, Established protocols can with collection of handling and be adopted for use in ecological data. preserving, collections. Investigators Develop protocol transport, apply protocols. for collecting, recording, handling and vouchering of preserving, specimens. transport, recording, etc. of specimens. 2. Establish Colonies of the Colony reports. Materials of all colonies of all recognized morphotypes can be morphotypes to morphotypes obtained and colonies allow established and established. behavioural, available for chemical, studies that cytological, larval required live morphology, etc. material. studies. 3. Carry out Cross-mating and Reports of The procedures to reproductive post-zygotic behaviour and establish species limits 70

compatibility studies carried out mating are scientifically analyses as between compatibility tests. acceptable. required between morphotypes of International peer selected the A. fraterculus reviewed populations of complex under publications. named species – semi-natural, including cross- choice situations, mating studies on to caged trees under confirm/evaluate semi-natural, presence of choice situations. reproductive Record isolation. behavioural aspects.

4. Perform a) Sex a) Reports of a) The samples are chemical, pheromone and pheromones representative of the cytological, cuticular studies and true population and molecular hydrocarbons analyses. are processed genetic, profiles according to the morphological determined for established protocol. and other relevant recognized biological studies morphotypes. to help b) Cytological b) Karyotype and characterize/ studies polytene b) New gene regions can discriminate performed and chromosome be accessed and used to among karyotypes data published. screen populations. morphotypes. described for each of the recognized morphotypes. c) Molecular c) Reports and genetic published c) Sufficient analyses genetic representative carried out on analyses. samples available to material conduct appropriate collected from analysis. across the geographical range of A. fraterculus s. l . d) Morphological d) Reports and studies carried published d) The samples are out on material morphological representative of the collected from studies. true population and across the that they are geographical processed according range of A. to the established fraterculus s. l . protocol. e) Other e) Published data 71

potentially on biology of discriminating flies with e) Experimental staff biological particular capable of carrying attributes reference to out standardised investigated assisting behavioural for all discrimination protocols. morphotypes. between populations/species. 5. Describe and Morphotypes Published results on Independent avenues of diagnose described and the taxonomy of the conducted research will morphotypes by diagnosed based A. fraterculus suggest a consensus collating, on the information complex focussing outcome. analysing and collected using on species of The supporting research interpreting new different economic as part of CRP (mating, and existing data. approaches. significance. pheromones, behaviour, etc. studies) is published and/or made available. 6. Clarify the -Holotypes exa- Holotypes. Funding for Consultants nomenclature for mined / synonyms Meeting reports. Meeting. A. fraterculus s. l. resurrected as (If name changes appropriate. are required, the -Consultant Inter-national meeting of Commission of specialists held to Zoological review all Nomen-clature evidence collected may need to be to name/ rename involved.) species. -Species named/ renamed. 7. Develop Diagnostic tools Published The previous objective improved developed based diagnostic (defining species limits diagnostic tools on morphological, protocols. in A. fraterculus ) has for each newly molecular and been achieved. described/rename other evidence Having determined d species within collected. species limits, diagnostic the complex. markers can be found.

OUTPUTS B. dorsalis

1. Carry out Protocols Published protocol Centralised group co- targeted sampling developed and checklist publicly ordinating collection and for species listed network available. curation of samples. as Bactrocera established for Local collectors dorsalis s. l . structured Central repository accurately locate and Establish a collecting, of collection collect required samples laboratory handling and material curated and according to defined network for preserving, live cultures protocols. 72

structured transport, established and Specialists available for sampling (for recording, etc. of maintained. specimen identification rearing, morpho- specimens. prior to live culture logy, pheromone establishment. and genetic work). 2. Quantify field Within species Results reported/ Confirming that B. cage mating variation in mating published. dorsalis s.s. is a single behaviour of two behaviour species allows the or more documented for B. establishment of geographically dorsalis s.s. centralised rearing distinct B. facilities. dorsalis s.s. Individuals collected populations as a from targeted collection basis for localities are B. dorsalis subsequent s.s. based on initial comparative morphological studies within the identification. complex. Collaborators can be found to undertake collections. 3. Carry out cross- Cross-matings and Published results on Flies can be maintained species matings behaviour studies cross-mating under wildish conditions under semi- carried out studies. over minimum natural, choice between species of generations to produce situations using the B. dorsalis meaningful results. wild / wildish complex to There is staff available to flies as required. compute and undertake experiments Record record mating and maintain cultures. behavioural isolation indices. The timing between field aspects as per QC collections, cultures, and Manual. available staff will coincide. 4. Carry out Multiple Published results on Experimental staff multiple generation crosses multiple post- capable of carrying out generation carried out in B. zygotic studies. standardised protocols. crosses to record dorsalis complex post-zygotic which detail post- isolation on at zygotic mating least two effects. Bactrocera dorsalis s. l . species. 5. Determine sex Sex pheromone Published results on The volatiles of pheromone profiles pheromone profiles. Bactrocera dorsalis s. l . profiles for each determined for species can be obtained species with and each species. and are processed without prior according to established methyl eugenol protocols. consumption. 73

6. Establish relative Relative taxon Published results on Suitable fly specimens attraction of sensitivity to relative attraction of and specialists (including males of different methyl eugenol males to methyl specialist equipment) are Bactrocera determined. eugenol. available to analyse and dorsalis s. l . compare information. species to methyl eugenol. 7. Investigate other Other potentially Reported protocols Experimental staff potentially discriminating made publicly capable of carrying out informative biological available. standardised biological biological attributes Published data on and behavioural attributes for investigated for biology of flies with protocols. discriminating species. particular reference between species. to assisting Quality of outcomes discrimination highly dependent on between essential co-ordination populations/species. between groups. 8. Carry out genetic, Genetic, Published results of New gene regions can be cytogenetic and cytogenetic and genetic analyses. accessed and used to molecular molecular analyses Reports with screen populations. analyses on carried out for B. specific statement Sufficient representative material collected dorsalis , B. on whether genetic samples available to from across the papayae and B. data supports conduct appropriate geographical philippinensis separate/single analysis. range of species populations. species for B. of economic dorsalis s.l . importance.

9. Species status for Species status Published results on Independent avenues of Bactrocera defined for at least the taxonomy of the conducted research will dorsalis s. l . B. dorsalis , B. B. dorsalis complex suggest a consensus species of papayae , B. focussing on outcome. economic philippinensis, B. species of economic The previous research as importance newly invadens and B. significance. part of CRP (mating, defined based on carambolae. pheromones, behaviour) outputs 1 to 8. is completed and made available. 10. Develop At least one Diagnostic The previous objective improved diagnostic tool protocols published. (defining species limits morphological developed for each in B. dorsalis s.l. ) has and molecular species defined in been achieved. diagnostic tools output 7. Having determined based on the species limits, diagnostic acquired markers can be found. knowledge (for adults and immatures).

OUTPUTS Bactrocera cucurbitae 74

1. Develop protocol Protocol Protocol available Established protocols can for collecting, developed for to all investigators. be adopted for use in handling, structured collections. Investigators preserving, collecting, apply protocols. transport, handling and recording, etc. of preserving, specimens from transport, Africa, Hawaii recording, and and Indian vouchering. Ocean. 2. Establish a Clarification on Published results on Flies can be built up in network for existence or non- mating culture to sufficient targeted, existence of host compatibility, numbers over minimum structured races within B. ecology and generations to produce sampling of cucurbitae among population genetics. meaningful results. species together populations from There is staff available to with ecological Africa, Hawaii and undertake experiments data. Indian Ocean. and maintain cultures. The timing between field collections, cultures, and available staff coincides. 3. Characterize Potential host Reports and Host races obtained and potential host races characterized publications of the presence of host races races through and differences ecological , is confirmed. ecological, explored. morphometric and morpho-metric, genetic studies. genetic studies. If there are positive differences then pro-ceed to other studies. 4. Carry out cross- Mating compati- Mating test reports Extent of cross-mating matings as bility assessments and publication. tests dependent on results required between and behaviour of characterization of selected studies carried out potential host races populations under among populations through ecological , semi-natural, from Africa, morphometric and choice situations Hawaii and Indian genetic studies. using wild / Ocean, under wildish flies. semi-natural, Record choice situations behavioural using wild / aspects. wildish flies.

OUTPUTS C. rosa , C. fasciventris, C. anonae

1. Develop Robust Markers to analyse More robust markers can morphological discriminating extensive field be identified and utilized. and molecular markers developed samples. diagnostics to be and made able to available for the distinguish the recognized recognized entities. entities. 2. Evaluate of Additional support Reports and Confirmation depending specific status of to the current or publications of on outcome o all other taxa within the changed specific taxonomic and studies. complex (e.g. status of taxa related studies. molecular, within the FAR pheromone, complex. cuticular hydrocarbon, mating compatibility, ecology, etc.)

ACTIVITIES

Hold Consultants Consultants CRP proposal. CRP approved by Meeting. meeting held 6-10 committee July 2009. Proposals submitted/accepted. Award research Research contracts Signed agreements Agreements and agreements and agreements. and contracts. contracts approved and contracts to fruit fly funded. researchers, taxonomists and fruit fly control programmes and establish CRP.

Organise 1 st RCM to 1st RCM held in Working material Consultants’ report plan, co-ordinate and August 2010. of 1 st RCM printed refined by participants. review research and distributed. activities.

Carry out R and D. R and D findings. Research reports. Continued institutional support, including support for establishment of colonies. Hold 2 nd RCM to 2nd RCM held in Working material Analytical tools review results, begin February 2012. printed and appropriate and analysis of collection distributed for 2 nd verifiable. data, draft technical RCM, research protocols as required, published in 76 and plan future scientific literature activities. and disseminated to Member States and Scientific community.

Continue R and D. R and D findings. Research reports. Continued institutional support. Hold training course Training course Trained A. Availability of lecturer(s) on A. fraterculus held in 2012. fraterculus and of funding. morphotype taxonomists in identification. region. Hold 3 rd RCM to 3rd RCM held in Working material Reports published and review results, June 2013. printed and distributed following continue analysis of distributed for 3rd each RCM. collections and RCM, research Mid-year review of CRP colony material and published in approved. to plan future scientific literature activities. and disseminated to Member States and Scientific community.

Hold Consultants Consultants Consultants Funding and Meeting of Meeting held, gasp Meeting report. participation of relevant specialists to review identified and specialists. all evidence collected decisions on some to review species species to be limits and named/ renamed. name/rename species.

Carry out final R and R and D findings. Research reports. Continued institutional D. support.

Hold final RCM to Final RCM held in Final CRP report Funding available to review data, reach January 2015. distributed and complete CRP and to consensus and research published publish proceedings. prepare final report. in scientific literature.

F. Workshop on “Reviewing Evidence to Resolve Species Complexes of Tephritid Pests” (31. August 2013)

Recommendations of External Panel

31 August 2013 (Agenda of Workshop in Annex 4)

1. Anastrepha fraterculus Complex Work Group

(see Current Knowledge of Anastrepha fraterculus Complex in Annex 1a and Character Matrix for the Anastrepha fraterculus Species Complex in Annex 1b )

Strengths: • Well-defined morphometric framework for differentiating the 7-8 morphotypes currently recognized. • Multiple lines of evidence in support of conclusions for separating various subsets of morphotypes as significantly different entities (e.g. pre- and post-zygotic isolation, cytology, cuticular hydrocarbons). • Extensive sampling of some morphotypes across range. • Extensive network of collaborators across a diverse range of disciplines.

Weaknesses: • Number of project participants contributing to morphometric identifications limited. • Molecular data available for study is insufficient, and would be highly desirable. • Molecular priorities unclear and how these relate to overall goals. • Movement of material between participants is highly restricted (legislative). • Different studies are frequently using only subsets of morphotypes, rather than comparing all 8 morphotypes. • Inadequate communications between workers doing molecular work. • Insufficient funds for sampling, molecular and morphometric work.

Recommendations (CRP): • Prioritization of morphological identification of Brazilian populations within current morphometric framework. • Ensure accurate morphotype identification using morphometric methods is made on all samples as a first step before use in other studies. • Prioritize objectives for molecular markers and analytical methods (phylogenetics, haplotype networks, etc.). • Work plan should ensure that all morphotypes are included in all studies, rather than subsets. 78

• Attempt to collect specimens from Venezuela, Ecuador, Andean Peru and Bolivia and establish colonies in Siebersdorf lab. • Trade implications should be considered, including communications strategy. • Develop diagnostic tools for all life stages. • IAEA and other relevant agencies be engaged in facilitating movement of samples and alleviating legislative restrictions. • Ensure samples are vouchered and availability where possible for future molecular studies.

Future recommendations:

• Where possible maintain IAEA colonies used for studies or voucher specimens • Cryopreserve IAEA colonies, if possible, for use in future studies.

2. Bactrocera dorsalis Complex Work Group

(see Current Knowledge of Bactrocera dorsalis Complex in Annex 2a and Character Matrix for the Bactrocera dorsalis Species Complex in Annex 2b )

Strengths: • Extensive sampling over entire distribution • Ready availability of material to participants throughout project duration • Coordination of participant roles and alignment of activities with project goals resulting in direct comparability of data and results • Clearly defined hypotheses • Clear presence of outlying groups such as B. occipitalis , and B. cacuminata • Multiple lines of evidence (i.e. morphometric, DNA sequences, mating behavior, pheromones, etc.) in support of conclusions of synonomy of B. papayae, B. philippinensis and B. invadens with B. dorsalis but not B. carambolae • Well-defined plan for publication of results with inclusive authorship.

Weaknesses: • Vouchering and likely availability of samples for future molecular studies.

Recommendations (CRP): • • Manuscript on synonymy of various species under B. dorsalis should include revised diagnosis and redescription of B. dorsalis s.s. • Trade implications should be considered, including communications strategy. • Consider longer term storage solutions for voucher specimens. • Develop diagnostic tools where applicable and available for all life stages. • Ensure samples are vouchered and availability where possible for future molecular studies

Future recommendations: • Further investigation of possible hybridization between B. carambloae and dorsalis complex. 79

• Clarification of potential issues of paraphyly of B. dorsalis relative to B. kandiensis in Sri Lanka and India. •

3. Ceratitis FAR Complex Work Group

(see Current Knowledge of Ceratitis FAR Complex in Annex 3a and Character Matrix for the Ceratitis FAR Species Complex in Annex 3b )

Strengths: • Extensive sampling over entire distribution • Ready availability of material to participants throughout project duration • Coordination of participant roles and alignment of activities with project goals resulting in direct comparability of data and results • Clearly defined hypotheses • Evidence in support of conclusions is relatively strong for FAR and R1 vs R2 differentiation, and based on adult male morphology, mating behavior [? Clarification of ISI results ], microsat and phys data. • Prioritizing work-plan and project activities based on relative economic importance of C. rosa and likely availability of material (i.e. rarity of C. fasciventris (West)).

Weaknesses: • DNA diagnostic tools for differentiating morphotypes and species are relatively weak and requires development.

Recommendations (CRP): • Trade implications should be considered, including communications strategy including NPPO communications. • Develop molecular and morphological diagnostic tools for all life stages. • Ensure samples are vouchered and available where possible for future molecular studies.

Future recommendations: • Continue to consider other morphological differences between species and morphotypes, and relationship to distribution. • Reanalysis of previously published ecological niche modeling data to predict potential distributions. • Further exploration of F1 – F2 differentiation.

Annex 1a: CURRENT KNOWLEDGE OF ANASTREPHA FRATERCULUS COMPLEX (August 2013)

The concept that Anastrepha fraterculus is a complex of cryptic species is not recent (Stone 1942, Morgante et al . 1980, Solferini & Morgante 1987, Malavasi & Morgante 1982, Steck 1991, Steck & Sheppard 1993, Selivon 1996, Hernández-Ortíz et al. 2004, 2012) and is based on the presence of population differentiation and reproductive isolation. The combined results of these studies, suggest the existence of at least seven different biological entities which are referred to as: A. sp. 1 aff. fraterculus (Yamada & Selivon 2001), A. sp. 2 aff. fraterculus (Yamada & Selivon 2001), A. sp. 3 aff. fraterculus (Selivon et al . 2004), A. sp. 4 aff. fraterculus (Selivon et al. 2004), A. fraterculus Mexican morphotype (Hernández-Ortíz et al . 2004) and A. fraterculus from Andean highlands and Venezuela coastal lowlands (Steck 1991). A recent comprehensive morphological study supports the existence of these seven morphotypes (Hernández-Ortíz et al. 2012). In order to determine if the biological entities correspond to different species, it was agreed for this CRP that there was a need to use different methodologies to analyse them, to use properly identified material, and most importantly, to include all of the morphotypes in the studies. This approach was followed since the initiation of the CRP and during the last months, significant new information appeared (see following summary character matrix for the Anastrepha fraterculus complex). At the molecular level, SSR markers were developed and applied to assess intra/inter- population differentiation (Malacrida). In addition, a portion of the cytochrome oxidase c subunit I was sequenced for several samples obtained from different populations from Brazil, Argentina and Mexico (Silva). Results indicate the presence of population structuring. Studies of COI and COII done with nine populations from Colombia showed the occurrence of the highland morphotype in eight of the populations with the ninth population, collected in the south, clustering apart (Canal). Preliminary DNA sequence data from Peru showed differences between populations from Cusco Department and coastal populations, as well as from those from eastern slopes of the Andes in Bolivia, which match those of Argentina and southern Brazil (Steck). Morphometric studies incorporated the geometric approach and new populations from Ecuador and results allow preliminary interpretations suggesting the occurrence of an additional eighth morphotype (Hernández-Ortiz). In addition, samples from adults collected from traps and larvae obtained from 14 different host plants in Peru allowed obtaining material for taxonomic analysis from regions in which material was scarce. This study is ongoing. In terms of behavior, studies keep providing evidence in support of the usefulness of field cages as a tool to determine mating compatibility among populations. As a result, it was possible to increase the number of population combinations analyzed and delineate better the degree of compatibility among them. The presence of full compatibility among populations from southern Brazil was confirmed, the existence of incompatibility between these populations and the population from Piracicaba (southeast Brazil), and contrary to expectations, the population from the northeast was compatible with one from the south (Joachim-Bravo). At the continental level, prezygotic isolation was revealed even when sexual activity occurred at the same time such as in the case of Mexico and Brazil 1 morphotypes (Rull). The addition of acoustical analysis allowed extending the tools available to analyze sexual isolation (Joachim-Bravo). 81

At the chemical level, it was evidenced that pheromones can act as a primary method of reproductive isolation. This is supported by the differences found in the volatiles analyzed from the different populations (Brizova, do Nascimiento, Teal) as well as for the cuticular hydrocarbons (Vanickova). In addition the incorporation of the chemical characterization of the proteins from the males’ accessory glands can provide another diagnostic tool (Rull).

Conclusions

− All evidence so far points towards the fact that previously reported population differences are correlated with reproductive isolation and if the biological concept of species is followed, Anastrepha fraterculus is composed of several species.

− Different tools can be used in combination for diagnosis (DNA, morphology, cytology, sexual behavior and chemical profile of male-emitted volatiles and cuticle extracts).

− All reproductively isolated populations revealed prezygotic isolation.

− As more populations/areas are incorporated into the comprehensive approach, the idea of the existence of morphotypes gains support.

− As a consequence it would be advisable to initiate progressively the process of species definition and revision.

Annex 1b: Character Matrix for the Anastrepha fraterculus Species Complex

A. sp. 1 A. sp. 2 A. sp. 3 Ecu Parn aff. aff. aff. Bentos Charac- Peru- Ande Mexi- Vene- a- Piraci- a- fratercu fratercu fratercu Gonçal ters vian -an can zuelan dori caba miri lus lus lus -ves an m

1. DNA- CO I

2. DNA - ITS1 If

and possible ITS2 3. DNA - CO II

4. Isozy- mes

5. Micro sa- telli- Done tes validat ion 6. Micro sa- telli- tes population analysis 7. Cytolo P P P P P gy 8. Adult mor- P P P P P P P pholo gy 9. Wing geo-

metric s 10. Lar- val morpho- logy 11. Egg morpho- P P ? ? logy

12. Pre- zygo- Partial tic P P P isolatio isola- n tion 83

13. Post- zygo- Bento Piracic tic P P P P Gonçal aba isolati ves on 14. Phero P P mo- nes 15. Cuticu lar P hydro carbon s 16. Remat ing behavi our 17. If If Sexual P possi possibl behavi ble e or 18. Time A. sp. 1 sexual A. sp. 1 aff. Piracica activit aff. fratercu ba / y fratercu lus / Mexican lus Mexica n 19. Sound

20. Host range P P P

21. Strain Vacaria (on / Pelotas Piura artifi- / Sao + La Toli- Xalapa cial Joaquin Moli- ma diet) / Tucu- na mán

Not a Differs Publi publicat from - ion that some shed reduces morpho Diffe- In but Norr- -type/s Legend rent to progr need bom's and all ess s to host over- be range laps upda with -ted other/s

1. -- 2. -- 3. -- 4. Criteria to distinguish from other populations based on Roger's Genetic Distance equal to or greater than distance between other recognized species of A. fraterculus group ( A. distincta and A. obliqua ). 5. Work developed in conjunction with INTA Buenos Aires and Pavia. 84

6. Not done yet. 7. Based on Selivón´s work and Cáceres et al. 2009; all morphotypes with different caryotype. 8. Based on Hernández-Ortiz et al. 2012; the Ecuadorian morphotype is not published. 9. Preliminary results show that geometric analysis can differentiate the eight morphotypes. 10. -- 11. Eggs show morphological differences. 12. All based on field cage mating compatibility tests. Males and females of two populations released in the cage. Mating compatibility indices range from high isolation (above 0.8), partial isolation (between 0.4 and 0.6) and random mating (below 0.2. All populations' combinations within a given morphotype show random mating (sp. 1, Peru, Colombia and Mexico). 13. Reduction in egg hatch and sex ratio distortion; published by Selivón and Cáceres. 14. All populations analysed present a different chemical profile, even some belonging to the same morphotype except Bento Gonzalvez and Piracicaba that showed the same chemical profile. Hybrids between Peru and sp. 1 present components unique to both populations (the blend is a mixture). Hybrid females prefer hybrid males. 15. As for pheromones, sensitivity seems higher than morphometrics. 16. Based on the remating rate of the females, the time between 1 st and 2 nd mating and the percentage of females without sperm transfer; manuscript submitted. 17. Based on video recordings; one publication for one Argentina population (Gomez- Cendra). 18. Some populations are temporarily isolated as they are early morning, midday and dusk maters. 19. Preliminary data that shows differences in the frequency. 20. Taking into account Norrbom's host list, Colombia and Mexico morphotypes have been claimed to have a narrower host range than Af ss. 21. Names of the localities from where populations were derived.

Annex 2a: CURRENT KNOWLEDGE OF BACTROCERA DORSALIS COMPLEX (August 2013) Background The Bactrocera dorsalis species complex was first recognised by Hardy (1969), and greatly expanded by Drew and Hancock (1994). Several other taxa have been placed in the complex outside of these two revisions, of which the only one of current relevance is B. invadens (Drew et al. 2005). While the majority of species within the B. dorsalis complex are easily diagnosed on morphological characters, this is not the case for a small group of cryptic taxa which have been the focus of the current project: B. dorsalis s.s. (Hendel), B. papayae Drew & Hancock, B. philippinensis Drew & Hancock, B. carambolae Drew & Hancock and B. invadens Drew, Tsuruta & White. Quite quickly after their original descriptions, concerns were raised as to the biological validity of B. papayae, B. carambolae and B. philippinensis, most notably by Prof Keng Hong Tan and colleagues (Nishida et al. 1988, Tan & Nishida 1996), but these views were subsequently reviewed and dismissed (Clarke et al. 2005, Drew et al. 2008). Nevertheless, repeated failure to find robust and consistent biological markers for these taxa (with the possible exception of B. carambolae ), has meant that the question of whether the taxonomic species correctly align with the biological species has never been fully resolved. This component of the CRP was established to (i) accurately delimit the biological species of this clade; and (ii) having done so develop appropriate diagnostic markers. Current status Very significant research has been undertaken, within an integrative taxonomic framework, to biologically delimit the target taxa (see following summary character matrix). The tools used have included: i) morphological examination, including traditional morphology, morphometrics of genitalic characters, and fine-scale wing shape analysis using geometric morphometrics; ii) molecular-based approaches using both phylogenetic and population genetic analyses of nuclear DNA sequence and microsatellite data coupled with mitochondrial DNA sequence analysis and comparative transcriptome analysis; iii) studies of mating and post-zygotic sexual compatibility; iv) analysis of rectal gland (= pheromone) constituents; and v) comparative polytene chromosone analysis. Not all work has yet been published, but much has (Tan et al. 2011, Khamis et al. 2012, Schutze et al. 2012a, Schutze et al. 2012b, Boykin et al. 2013, Krosch et al. 2013, Schutze et al. 2013, Tan et al. 2013). With one exception, all data so far presented by CRP participants have failed to find evidence of any species level biological differentiation between B. dorsalis , B. papayae , B. philippinensis and B. invadens . Any genetic and morphological differences which have been found are best explained as intra-specific variation (i.e. population level variation), not inter- specific variation. Data (pheromonal, genetic, behavioural) suggests that B. carambolae is a biologically unique, albeit very closely related species to the other four taxa. It is important to recognise that some critical data sets for B. invadens (e.g. sexual compatibility trials) have still to be publically presented, but this ‘missing’ data will be presented in Argentina and is consistent with the other data sets so far presented. The one exception to the general patterns seen in the data sets was a verbal paper presented by Prof Drew at the 2nd RCM (Brisbane, Jan 2012), who analysed thoracic colour patterns of B. invadens from Africa and B. dorsalis from China and concluded that the frequency of colours (predominantly red-brown in the African flies and black in the Chinese flies) confirmed these two species as described. Because such colour variation might also be interpreted as intra-specific variation in one wide-ranging biological species, more work in this area has been done in the last 18 months and will be presented in Argentina. 86

Prof Drew has recently formally synonymised B. philippinensis with B. papayae in a soon to be published monograph, but no other taxonomic changes have been made.

Conclusions • All evidence so far suggests that B. dorsalis , B. papayae and B. philippinensis are one biological species, while B. carambolae is a closely related but biologically unique species, albeit natural hybrids between B. carambolae and B. papayae have been detected in the Penisular Malaysia (Tan, 2005, Wee and Tan 2005). • B. invadens should also be likely considered a junior synonym of B. dorsalis , but some additional data sets need to be publically presented and peer considered before this can be assumed. • The strength of the case for considering the synonymisation of B. papayae, B. philippinensis and B. invadens with B. dorsalis rests not on the strength of any one approach (i.e. mating, pheromone, genetics), but the highly consistent results of all of these independent approaches.

References Boykin, L. M., M. K. Schutze, M. N. Krosch, A. Chomic, T. A. Chapman, A. Englezou, K. F. Armstrong, A. R. Clarke, D. Hailstones, and S. L. Cameron. 2013. Multi-gene phylogenetic analysis of the south-east Asian pest members of the Bactrocera dorsalis species complex (Diptera: Tephritidae) does not support current taxonomy. Journal of Applied Entomology:DOI: 10.1111/jen.12047. Clarke, A. R., K. F. Armstrong, A. E. Carmichael, J. R. Milne, S. Raghu, G. K. Roderick, and D. K. Yeates. 2005. Invasive phytophagous pests arising through a recent tropical evolutionary radiation: The Bactrocera dorsalis complex of fruit flies. Annual Review of Entomology 50 :293-319. Drew, R. A. I. and D. L. Hancock. 1994. The Bactrocera dorsalis complex of fruit flies (Diptera: Tephritidae: Dacinae) in Asia. Bulletin of Entomological Research Supplement No. 2 :i-iii, 1-68. Drew, R. A. I., S. Raghu, and P. Halcoop. 2008. Bridging the morphological and biological species concepts: studies on the Bactrocera dorsalis (Hendel) complex (Diptera: Tephritidae: Dacinae) in South-east Asia. Biological Journal of the Linnean Society 93 :217-226. Drew, R. A. I., K. Tsuruta, and I. M. White. 2005. A new species of pest fruit fly (Diptera: Tephritidae: Dacinae) from Sri Lanka and Africa. African Entomologist 13: 149-154. Hardy, D. E. 1969. Taxonomy and distribution of the oriental fruit fly and related species (Tephritidae-Diptera). Proceedings of the Hawaiian Entomological Society 20 :395- 428. Khamis, F. M., D. K. Masiga, S. A. Mohamed, D. Salifu, M. d. Meyer, and S. Ekesi. 2012. Taxonomic identity of the Invasive fruit fly pest, Bactrocera invadens : Concordance in morphometry and DNA barcoding. PLoS ONE:7(9) e44862. doi:44810.41371/journal.pone.0044862. Krosch, M. N., M. K. Schutze, K. F. Armstrong, Y. Boontop, L. M. Boykin, T. A. Chapman, A. Englezou, S. L. Cameron, and A. R. Clarke. 2013. Piecing together an integrative taxonomic puzzle: microsatellite, wing shape and aedeagus length analysis of Bactrocera dorsalis s.l. (Diptera: Tephritidae) find no evidence of multiple lineages in a proposed contact zone along the Thai/Malay Peninsula. Systematic Entomology 38 :2-13. 87

Nishida, R., K.H. Tan, M. Serit, N.H.Lajis, A.M. Sukari, S. Takahashi and H. Fukami. 1988. Accumulation of phenylpropanoids in the rectal glands of male Oriental fruit fly, Dacus dorsalis . Experientia 44 :534-536. Schutze, M., A. Jessup, I. Ul-Haq, M. J. B. Vreysen, V. Wornoayporn, M. T. Vera, and A. R. Clarke. 2013. Mating compatibility among four pest members of the Bactrocera dorsalis fruit fly species complex (Diptera: Tephritidae). Journal of Economic Entomology 106 :695-707. Schutze, M. K., A. Jessup, and A. R. Clarke. 2012a. Wing shape as a potential discriminator of morphologically similar pest taxa within the Bactrocera dorsalis species complex (Diptera: Tephritidae). Bulletin of Entomological Research 102 :103-111. Schutze, M. K., M. N. Krosch, K. F. Armstrong, T. A. Chapman, A. Englezou, A. Chomič, S. L. Cameron, D. L. Hailstones, and A. R. Clarke. 2012b. Population structure of Bactrocera dorsalis s.s., B. papayae and B. philippinensis (Diptera: Tephritidae) in southeast Asia: evidence for a single species hypothesis using mitochondrial DNA and wing-shape data. BMC Evolutionary Biology 12 :130. doi:110.1186/1471-2148- 1112-1130. Tan, K. H. 2005. Interbreeding and DNA analysis of Bactrocera dorsalis sibling species. pp 113 – 122. In: Recent Trends on Sterile Insect Technique and Area-Wide Integrated Pest Management - Economic Feasibility, Control Projects, Farmer Organization and Bactrocera dorsalis Complex Control Study . Research Institute for the Subtropics, Okinawa, Japan. Tan, K. H. and R. Nishida. 1996. Sex pheromone and mating competition after methyl eugenol consumption in Bactrocera dorsalis complex. pp. 147-153. In: B. A.McPheron and G.J. Steck (eds.) Fruit Fly Pests – A World Assessment of their Biology and Management. St.Lucie Press, Florida. Tan, K. H., I. Tokushima, H. Ono, and R. Nishida. 2011. Comparison of phenylpropanoid volatiles in male rectal and pheromone gland after methyl eugenol consumptions, and molecular phylogenetic relationship of four global pest fruit fly species: Bactrocera invadens, B. dorsalis, B. correcta and B. zonata . Chemoecology 21 :25-33. Tan, K. H., S.L. Wee, H. Ono, and R. Nishida. 2013. Comparison of methyl eugenol metabolites, mitochondrial COI, and rDNA sequences of Bactrocera philippinensis (Diptera: Tephritidae) with those of three other major pest species within the dorsalis complex. Applied Entomology and Zoology 48 :275-282. Wee, S. L. and K. H. Tan. 2005. Evidence of natural hybridization between two sympatric sibling species of Bactrocera dorsalis complex based on pheromone analysis. Journal of Chemical Ecology 31 :845-858.

Annex 2b: Character Matrix for the Bactrocera dorsalis Complex

Characters B. dorsalis s.s. B. papayae B. philippinensis B. carambolae B. invadens B. kandiensis B. occipitalis

1. Polytene chromosomes 2. Rectal gland extracts following ME feeding 3. Mechanics of processing of ME 4. Thoracic colour pattern 5. Aculeus spicules

6. Pre-zygotic compatibility 7. Post-zygotic compatibility 8. Multi-gene phylogeny

9. CO1 haplotype (pop genetics) 10. Microsatellite (pop genetics) 11. Wing shape

12. Y chromosome

13. Post-suttural lateral vittae 14. Aedeagus length 15. Cuticular hydrocarbons 16. General morphology

No differences Differences found Legend to other taxa to the other taxa

1. "The complex is homosequential", but with some minor inversions in B. dorsalis vs. B. carambolae hybrids. 2. The constitutive pheromone compounds and ME derived metabolites are the same for B. dorsalis, B. papaya, B. philippinensis and B. invadens , but different not for B. carambolae. 3. The physiological processing of ME seems the same for B. dorsalis, B. papayae, B. philippinensis and B. invadens , but different for B. carambolae . 4. B. invadens is predominantly red-brown and the others black, but it has been shown that thoracic colour is a qualitative genetic trait, which exhibits at different population frequencies in different locations. 89

5. Originally thought to be discriminating between B. papayae and B. philippinensis , subsequently shown not to be in a publication by Khalid Mahmood. 6. In standardised field cage mating trials, random mating shown between B. dorsalis, B. papayae, and B. philippinensis , but not B. carambolae . In a separate set of tests, B. invadens was shown to mate randomly with two B. dorsalis populations (China and Pakistan). 7. Compatibility shown to F 2 generation. Non-compatibility shows up as significantly reduced egg hatch in the F 1 population. 8. Bayesian phylogeny using 2 mitochondrial, 2 nuclear and 2 ribosomal genes. 9. Co1 haplotype networks tightly clustered for studies of B. dorsalis, B. papaya, B. philippinensis and B. invadens . B. carambolae and B. kandiensis share no haplotypes with this cluster (or each other). 10. Microsatellites detect no differences in B. dorsalis and B. papayae in the Thai/Malay Peninsula where these two taxa are thought to abut or overlap their distributions. 11. Wing shape between B. dorsalis, B. papayae and B. philippinensis variable, but showing very strong isolation by distance effects consistent with a single species hypothesis. B. invadens wing shape very similar to B. dorsalis populations from northern India/Pakistan. 12. --- 13. Originally thought to be discriminating between B. dorsalis and B. invadens , subsequently shown to be a clinal trait. Not different between B. dorsalis and B. papayae . 14. Originally thought to be discriminating between B. dorsalis and B. papayae , subsequently shown to be a clinal trait. Not different between B. dorsalis and B. invadens . 15. Work done on cuticular hydrocarbons for these flies on-going. 16. B. carambolae, B. kandiensis and B. occipitalis relatively straightforward to separate on subtle, but relatively consistent morphological characters. B. dorsalis, B. papaya, B. philippinensis and B. invadens cannot be consistently separated in this way.

Annex 3a: CURRENT KNOWLEDGE CERATITIS FAR COMPLEX (August 2013)

The Ceratitis FAR complex comprises three described entities: C. rosa Karsch, 1887 C. rosa var. fasciventris (Bezzi, 1920) C. anonae Graham, 1908

C. rosa var. fasciventris was given specific status by De Meyer (2001). All three entities are thus currently recognized as separate species. Males can be easily differentiated, based on secondary male characters (pilosity of the mid leg. See De Meyer & Freidberg, 2006). Females cannot be easily differentiated. There are some minute differences in leg and anepisternal pilosity between C. anonae and the other two species (cf De Meyer & Freidberg, 2006).

However, no mitochondrial or nuclear markers were found so far to serve as unambiguous differentiating character between the three entities (Barr & McPheron, 2006; Virgilio et al, 2008; Barr & Wiegmann, 2009). Also in laboratory conditions, C. rosa and C. fasciventris can interbreed (G. Franz, pers. communication) although the hybrids show a higher fluctuating asymmetry which might indicate a decrease in developmental stability (Erbout et al, 2008). Therefore, it is not clear whether the three entities correspond to true biological species.

Pheromone and cuticular hydrocarbon analyses showed that the three entities are clearly separable based on these methodologies (2 nd RCM meeting report and presentations; Vacinkova et al, subm.). Morphology of immature stages also showed differences specimens of C. rosa (2 nd RCM meeting report and presentation by G. Steck), while the use of morphological characters to separate the three entities needs further examination. Recent morphometric research, based on wing landmarking, also allows separation of the three entities (3 rd RCM meeting presentation De Meyer).

Using microsatellite markers, it was shown that the three entities could be recognized but that both C. rosa and C. fasciventris each consist of two separate genotypes (R1-R2 and F1-F2) resulting in five different entities within the complex.

Recent work has focused on re-analyzing existing data and extending previously used methodologies recognizing the five entities:

C. anonae is a single entity and is not further investigated. Both males and (albeit to a lesser degree) females can be differentiated from the other four entities. It shows a clear geographic pattern unlike that of the other entities (although with some considerable overlap).

Research on C. fasciventris is restricted to re-analyzing the adult morphology, host plant and geographic data. Male adults can be differentiated by characters of the mid tibia. Wing landmarking gives an indication of separation for both sexes but not significantly. The data on host plants are too limited to make draw any conclusions on possible differences in host spectrum. The distributional data show two largely separated geographical occurrences with one type (F2) restricted to eastern Africa, while the other type (F1) mainly found in western and central Africa (this separations was also confirmed by the markers used in Virgilio et al., 2008). However the latter type is also found in some isolated areas in Angola, Malawi, Tanzania and Zambia. No clear geographical or other pattern could be detected. The lack of 91 an established colony of both types has prevented providing material for some of the other studies (like larval morphology or pheromones) and did not allow checking on mating incompatibility and physiological differences. Currently there is an established colony of F2 type (from eastern Africa) at ICIPE (Kenya). Contacts have been made with researchers in western Africa but establishment of live colony of F1 has been unsuccessful so far.

Most research has been focusing on the two C. rosa types, also because C. rosa is considered economically the most important species within the complex. Adult males can be differentiated, based on characters of the male mid tibia. Wing landmarking gives an indication of separation for both sexes but not significantly. There is conflicting information in the literature regarding developmental physiology and climatic niche for C. rosa (De Meyer et al, 2008; Duyck et al., 2002, Grout & Stoltz, 2007). Re-analysis of distributional data indicates that this might be because of failure to recognize the two types that were indicated by the microsatellite study. One type, R2, appears to occur at lower latitudes on the African continent and at higher altitudes. It might be more cold resistant than R1 type that is absent from the colder parts (lower latitudes, higher altitudes) within the geographic range of C. rosa . In order to corroborate this with experimental data, physiological development experiments are set up by different teams throughout the range of the species (South Africa, Tanzania and Kenya). So far live colonies of both types have be established only in Kenya by ICIPE. South African and Tanzanian teams are currently setting up live colonies. Preliminary results on the Kenyan experiments will be presented at the 3 rd CRM. In addition, material from the colonies at ICIPE has been made available to other teams for study of larval morphology and cuticular hydrocarbons. Preliminary investigation of larval morphology between R1 and R2 from Kenya has not shown any differences (but there is marked difference with earlier studied material of C. rosa (type unknown) from South Africa. Further results will be presented at the 3 rd CRM. Mating compatibility experiments have been conducted between the two C. rosa (R1-R2) and the C. fasciventris (F2) populations at ICIPE. These results will also be presented at the 3 rd CRM.

Conclusions - All evidence so far points towards the fact that the three recognized species within the FAR complex are indeed three separate species - Further evidence indicate that C. rosa and C. fasciventris might each comprise two different entities, while C. anonae remains a single entity - For the two C. fasciventris types, there is only the indication of some adult morphological differences and different biogeographic patterns. The latter are not completely understood so far. Further investigation in the status of the two entities will require the establishment of live colonies. - For the two C. rosa types, there is some evidence that these are indeed two separate species. Results presented at the 3 rd CRM and further research that is planned in the forthcoming months might further reveal the exact status of these two entities.

Annex 3b: Character Matrix for the Ceratitis FAR Complex

Discipline C. rosa 1 C. rosa 2 C. fasciventris 1 C. fasciventris 2 C. annonae Remarks (R1) (warm) (R2) (cold) (F1) (west) (F2) (east) (A) 1. Microsatellite (pop genetics) published 2. ND6/COI published 2. Per/ITS1 published 2. ITS2/other markers

3. Adult male morphology published 4. Adult female morphology published 5. Wing morphometrics dissertation 6. Larval morphology

7. Developmental physiology 7. Pre-zygotic isolation

7. Post-zygotic isolation

8. Pheromones dissertation 8. Cuticular hydrocarbons submitted 9. Host range

10. Distribution

11. Y-chromosome published 12. Endosymbionts

13. Behaviour

1. Recognition of five genotypes is based upon the microsatellite analysis. This splitting up was confirmed by male morphology. 2. Based on Virgilio et al. (2008). 3. Five types can be differentiated using secondary sexual characters of male mid femur and tibia ornamentation and shape. 4. Only female C. anonae can be differentiated from the other four types using pilosity characters on anepisternum and fore femur. 5. Male morphometrics allow some differentiation but not complete. 6. Preliminary studies on larval morphology do not show any distinct differences but need to be further examined in detail. 7. Study of C. anonae is deemed unnecessary given other means of differentiation. Current focus is on R1-R2 differentiation and needs to be elaborated. 8. Study done on three 'morphotypes' prior to splitting in five groups. Work will be extended to R1-R2. 9. All types are polyphagous and most data are too limited to observe distinct differences in host range, except for R1-R2 regarding temperate fruits. 93

10. All types show distinct distribution ranges. 11. Already published by Malacrida's team. 12. To be done on all 5 populations of the FAR ( Wolbachia presence). 13. Behavioural studies (including mating, responses to attractant etc.) with regard to the highland population of C. rosa collected from coffee (Ruiru) has previously been documented (Pontiano Nemeye’s thesis) but no information is available with regard to the lowland population. In addition to assessing mating compatibility, observation on courtship repertoire/mating behaviour will be recorded in field cages. Similarly, responses of the two populations to trimedlure, EGOlure and synthetic food attractants will be documented in field cages.

Annex 4: “Workshop on Reviewing Evidence to Resolve Species Complexes of Tephritid Pests”

31 August 2013

Tucumán Center Hotel, Tucuman, ARGENTINA

SATURDAY, 31 AUGUST

Chairperson: David Haymer

Panel of Independent Experts: Rob Duthie, David Haymer, Allen Norrbom, Shaun Winterton

08:00 - 08:15 David Haymer: Opening statements, goals of the workshop, review of the agenda and other issues.

08:15 - 08:30 Tony Clarke: Brief summary of CRP findings of the Bactrocera dorsalis complex group, focusing on conclusions and remaining controversies and research possible gaps.

08:30 - 09:00 Panellists: Questions and discussion to determine/confirm B. dorsalis complex: a. specifically what decisions need to be made by the end of the day; b. the areas where consensus has been reached and can be put aside.

09:00 - 09:15 Teresa Vera: Brief summary of CRP findings of the Anastrepha fraterculus complex group, focusing on conclusions and remaining controversies and possible research gaps.

09:15 - 09:45 Panellists: Questions and discussion to determine/confirm for the A. fraterculus complex: a. specifically what decisions need to be made by the end of the day; b. the areas where consensus has been reached and can be put aside.

09:45 - 10:00 Marc de Meyer: Brief summary of CRP findings of the Ceratitis FAR group, focusing on conclusions and remaining controversies and research possible gaps.

10:00 - 10:30 Panellists: Questions and discussion to determine/confirm for the Ceratitis FAR complex: a. specifically what decisions need to be made by the end of the day; b. the areas where consensus has been reached and can be put aside.

10:30 - 10:45 BREAK

10:45 - 11:30 Panellists: discussion on the controversies/gaps to debate, following two approaches: a. addressing a series of direct questions to the specific research groups; and b. guiding a semi-formal debate between representatives from those groups with differing opinions, mediated by the chair of the meeting.

12:00 - 14:00 LUNCH

Panellists discuss among themselves the arguments and formalise decisions as to whether: a. Sufficient data have been obtained to answer the remaining questions, in which case they deliver ‘verdicts’. b. In cases where remaining data are required to answer the questions, they state what remains to be done and suggests how this should be achieved.

14:00 - 14:30 Panellists: make a presentation to participants of all the groups on the outcome of their deliberations.

14:30 - 15:30 Panellists: chair a final discussion and guide reaching a consensus on follow-up actions by research groups during the final 18 month phase of the CRP.

15:30 - 15:45 BREAK

15:45 - 17:00 Panel lists: finalize recommendation and follow-up actions by research groups during the final 18 month phase of the CRP.

G. Appendices

Appendix 1: 3rd RCM Participants

LIST OF PARTICIPANTS 3rd RCM “Resolution of Cryptic Species Complexes of Tephritid Pests to Overcome Constraints to SIT Application and International Trade” Tucuman, Argentina 26-31st 2013

ARGENTINA

Mr BACHMANN, Guillermo Laboratorio de Genética de Insectos Instituto de Genética, INTA Castelar Nicolas Repetto y de los Reseros s/n (1686), Hurlingham Buenos Aires, ARGENTINA Email: [email protected]

Ms CERIANI CAMDESUS , Adriana Dirección de Cuarentena Vegetal SENASA Av. Paseo Colón 315, 4º piso, Oficina 14 Buenos Aires, ARGENTINA Tel: +54 11 4121-5245/5246 Email: [email protected]

Mr CLADERA , Jorge Instituto de Genética, INTA Castelar Nicolás Repetto y de los Reseros s/n (1686) Hurlingham, Buenos Aires, ARGENTINA Tel + 54 11 44501876 and 44598372 EXT 106 EMAIL [email protected]

Ms FEDYSZAK, Paola Yael Programa Nacional de Control y Erradicación de Mosca de los Frutos SENASA Av. Paseo Colón 315, 4º, Depto. A – Oficina 13 Buenos Aires, ARGENTINA E-mail: [email protected]

Ms GOANE , Lucia Facultad de Agronomía y Zootecnia UNT-Estación Experimental Agroindustrial Obispo Colombres (FAZ-EEAOC) Av. William Cross 3150-Las Talitas- Tucumán, ARGENTINA Tel: 0054 381 4521000 (243) Fax: 0054 381 4521000 (231) Email: [email protected]

Ms JOFRE BARUD , Flavia Instituto de Biotecnología Facultad de Ingeniería Universidad Nacional de San Juan Av. Libertador General San Martín 1109(O) San Juan, ARGENTINA Tel - Fax: 0264 4211700 Email: [email protected]

Ms JUAREZ , María Laura Cátedra Terapéutica Vegetal Facultad de Agronomía y Zootecnia Florentino Ameghino s/n. Finca El Manantial, Universidad Nacional de Tucumán Tucumán (4000), ARGENTINA Tel: (54) 381-439-0003 Email: [email protected]

Ms LOPEZ , María Liza Instituto de Biotecnología Facultad de Ingeniería Universidad Nacional de San Juan Av. Libertador General San Martín, 1109 (O) San Juan, ARGENTINA Tel - Fax: 0264 4211700 E-mail: [email protected]

Ms MENDOZA , Mariana Universidad Nacional de San Juan Av. Libertador General San Martín, 1109(O) San Juan, ARGENTINA Email: [email protected]

Mr MURUA , Fernando Programa de Control y Erradicación de Mosca de los Frutos Av. N. Benavides 8000 Oeste – Rivadavia San Juan, ARGENTINA Tel.-Fax: 0264 - 4231996 E-mail: [email protected] 99

Ms RUIZ , María Josefina CONICET- Estación Experimental Agroindustrial Obispo Colombres Av. William Cross 3150 - Las Talitas Tucumán, ARGENTINA Telefax 54-381-4521000-int.154 Email: [email protected]

Mr VILARDI Juan Universidad de Buenos Aires Buenos Aires, ARGENTINA Email: [email protected]

Ms VERA, María Teresa Universidad Nacional de Tucumán Avenida Independencia 1800 4000 San Miguel de Tucumán, ARGENTINA Tel: 0054 381 439 0003 Fax: 0054 381 4390049 Email: [email protected]

Ms VILLAGRAN , María Elvira Estación Experimental Agroindustrial Obispo Colombres Av. William Cross 3150 - C.P. T4101XAC - Las Talitas Tucumán, ARGENTINA Tel: (54 - 381) 452 1000 Email: [email protected]

AUSTRALIA

Mr CLARKE, Anthony Associate Professor Biogeosciences, FaST Queensland University of Technology G.P.O. Box 2434 Brisbane, QLD 4001 AUSTRALIA Tel: 61 7 3138 5023 Fax: 61 7 3138 1535 Email: [email protected]

Mr DUTHIE, Robert Plant Health Consultant PO Box 212 Bellingen, New South Wales 2454 AUSTRALIA Tel: (+61) 66551843 E-Mail: [email protected] 100

Mr SCHUTZE, Mark School of Earth, Environmental and Biological Sciences & CRC National Plant Biosecurity Queensland University of Technology, GPO Box 2434 Brisbane, QLD 4001, AUSTRALIA Tel: (Int) 61 7 3138 8351 Fax: (Int) 61 7 3138 1535 Email: [email protected]

AUSTRIA

Mr CÁCERES, Carlos Insect Pest Control Laboratory Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture FAO/IAEA Agriculture and Biotechnology Laboratories A-2444 Seibersdorf, AUSTRIA Tel.: +43 1 2600 28413 Email: [email protected]

Mr HAQ, Ihsan Insect Pest Control Laboratory Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture FAO/IAEA Agriculture and Biotechnology Laboratories A-2444 Seibersdorf, AUSTRIA Tel.: +43 1 2600 28454 Email: [email protected]

Mr HENDRICHS, Jorge Insect Pest Control Programme Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture Wagrammerstrasse 5, 1400 Vienna, AUSTRIA Tel.: +43 1 2600 21628 Email: [email protected]

BELGIUM

Mr DE MEYER, Marc Royal Museum for Central Africa Leuvensesteenweg 13 3080 Tervuren, BELGIUM Tel : 32 (0) 2 7695360 Fax : 32 (0) 2 7695695 Email : [email protected]

101

BRAZIL

Ms DE LIMA, Kátia Manuela Universidade Estadual de Santa Cruz Laboratório de Marcadores Moleculares e Citogenética Rodovia Ilhéus-Itabuna, km 16 BRAZIL Tel Lab: (0055 73) 3680-5431 Tel cel: (0055 73) 8801-4731 ou 8177-8558 Email : [email protected]

Ms GOMES DA SILVA MILLER, Janisete Centro de Biotecnologia e Genética Departamento de Ciências Biológicas Universidade Estadual de Santa Cruz Rodovia Ilhéus-Itabuna, km 16 BRAZIL Tel : 00557 3680 5440 Fax : 005573 3680 5226 Email : [email protected] [email protected]

Ms RUFINO DO NASCIMIENTO, Ruth Universidade Federal de Alagoas Av. Lourival Melo Mota, s/n Tabuleiro do Martins 57072-970 Maceio, BRAZIL Tel/Fax : 55 82 3214 1361 Email : [email protected]

Ms SORDI JOACHIM BRAVO, Iara Medfly Rearing Facility Brazil Quadra d-13, Iote 15 Distrito Industrial do Sao Francisco 48.900-000 Juazeiro, Bahia, BRAZIL Email: [email protected]

Ms PASSOS RORIZ , Kelly Empresa Baiana de Desenvolvimento Agrícola Laboratório de Ecologia Nuticional de Insetos Rua Barão de Geremoabo, Campus-Ondina-147, Instituto de Biologia Salvador, Bahia, BRAZIL Tel: 32836545 Email: [email protected]

102

COLOMBIA

Mr CANAL DAZA, Nelson Universidad del Tolima Barrio Santa Elena, Apartado Aéreo 546 Ibagué, Tolima, COLOMBIA Tel/Fax : 57 8 2771212, ext 9261. Email: [email protected]

Ms CASTAÑEDA, María del Rosario Universidad del Tolima Barrio Santa Elena, Apartado Aéreo 546 Ibagué, Tolima, COLOMBIA Tel/Fax : 57 8 2771212, ext 9261 . Email: [email protected]

CZECH REPUBLIC

Ms BRIZOVA, Radka Institute of Organic Chemistry and Biochemistry Academy of Sciences of the Czech Republic Flemingovo nám. 2, CZ-166 10 Praha 6 CZECH REPUBLIC Telephone: +420 220 183 240 E-mail: [email protected]

Ms KALINOVA, Blanka Institute of Organic Chemistry and Biochemistry Academy of Sciences of the Czech Republic Flemingovo nam.2, 166 10 Praha 6 CZECH REPUBLIC Tel: +420 220 183 339 Email: [email protected]

Ms VANICKOVA, Lucie Institute of Organic Chemistry and Biochemistry Academy of Sciences of the Czech Republic Flemingovo nam. 2, 166 10 Praha 6. CZECH REPUBLIC Tel: +420 220 183 339 Email: [email protected]

103

FRANCE

Ms DELATTE, Hélene UMR "Peuplements Végétaux et Bio-agresseurs en Milieu Tropical" CIRAD-3P 7, Chemin de l'IRAT Ligne Paradis 97410 Saint Pierre La Réunion, FRANCE Tel 262 262 49 92 35 Email: [email protected]

GREECE

Ms DROSOPOULOU , Elena Department of Genetics, Development and Molecular Biology School of Biology, Faculty of Sciences Aristotle University of Thessaloniki Thessaloniki, GREECE. Tel: 0030-2310 998291 (office) E-mail: [email protected]

Ms ZACHAROPOULOU , Antigone University of Patras 265 00 Patras, GREECE Email: [email protected]

ITALY

Ms MALACRIDA, Anna Dipartimento di Biologia Animale Via Ferrata 1 Università di Pavia 27100 Pavia, ITALY Tel/Fax +39 0382 986059 Email: [email protected]

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JAPAN

Mr TSURUTA, Kenji Pest Identification & Diagnostics Division, Yokohama Plant Protection Station, MAFF, 1-16-10, Shinyamashita, Naka-Ku, Yokohama, 231-0801 JAPAN TEL: +81-45-622-8940 FAX: +81-45-621-7560 E-mail: [email protected]

KENYA

Mr EKESI , Sunday International Centre of Insect Physiology and Ecology (ICIPE) P.O. Box 30772 Nairobi, KENYA Email: [email protected]

MALAYSIA

Mr HEE , Alvin University Putra Malaysia 43400 Serdang, Selangor, MALAYSIA Tel : 603 8946 6643 Email : [email protected]

Mr TAN, Ken Hong Phi Systems Sdn Bhd Phi Biotech Sdn Bhd (Research) 18-26-A1, Gurney tower Persiaran Gurney 10250 Penang, MALAYSIA Tel : 604 890537 Email : [email protected]

MEXICO

Mr HERNANDEZ-ORTIZ, Vicente Instituto de Ecología A.C. Carretera Antigua a Coatepec No. 35, El Haya Apartado Postal 63. CP. 91070 Xalapa, Veracruz, MEXICO Tel: 52 228 842 1800 ext 3303 Fax: 52 228 8421800 ext 4222 105

Email: [email protected]

Mr RULL, Juan Instituto de Ecologia A.C. Carretera Antigua a Coatepec No. 35, El Haya Apartado Postal 63. CP. 91070 Xalapa, Veracruz, MEXICO Email: [email protected]

NEW ZEALAND

Ms ARMSTRONG, Karen Bio-Protection Research Centre Lincoln University P. O. Box 84 Canterbury 8150, NEW ZEALAND Tel: 64 3 325 3838 Fax: 64 3 325 3864 Email: [email protected]

THAILAND

Ms AKETARAWONG, Nidchaya Department of Biotechnology Faculty of Science Mahidol University 272 Rama VI Road Ratchathewe, Bangkok 10400 THAILAND Tel: 668 9493 7036 Fax: 662 441 9820 ext 1124 Email: [email protected]

Ms CHINVINIJKUL, Suksom Irradiation for Agricultural Development Section Dept. of Agricultural Extension Ministry of Agriculture and Cooperatives (MOAC) Paholyotin Road, Chatujak, Bangkok 10900 THAILAND Tel: 662 5773682, 9406187 Fax: 662 5773682, 9406188 Email: [email protected]

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UNITED REPUBLIC OF TANZANIA

Mr MWATAWALA, Maulid Sokoine University of Agriculture P.O. Box 3000. Chuo Kikuu Morogoro, UNITED REPUBLIC OF TANZANIA Tel: + 255 23 260 3511 Fax: + 255 23 260 4651 Email: [email protected]

UNITED STATES OF AMERICA

Ms AGUIRRE HAYMER , Beatriz 454 Opihikao Pl. Honolulu, HI 96825. UNITED STATES OF AMERICA Email: [email protected]

Mr HAYMER, David Professor of Genetics Dept. of Cell and Molecular Biology 1960 East-West Rd, Biomed T511 University of Hawaii Honolulu, HI 96822. UNITED STATES OF AMERICA Email: [email protected]

Mr NORRBOM, Allen L. Systematic Entomology Lab., USDA c/o National Museum of Natural History, MRC-168 P.O. Box 37012 Washington, DC 20013-7012. UNITED STATES OF AMERICA Tel. 202 633-4564 Fax. 202 786-9422 Email: [email protected]

Mr STECK, Gary Division of Plant Industry Florida Dept. of Agriculture and Consumer Services P. O. Box 147100 Gainesville, FL 32614-7100. UNITED STATES OF AMERICA Tel: 1 352 372 3505 ext 188 Fax: 1 352 334 0737 Email: [email protected]

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Mr TEAL , Peter Chemistry Research Unit Center for Medical, Agricultural and Veterinary Entomology, USDA-ARS 1700 SW 23 Drive Gainesville, Fl. 32604. UNITED STATES OF AMERICA Email: [email protected]

Mr WINTERTON , Shaun California State Collection of Arthropods California Department of Food and Agriculture 3294 Meadowview Rd. Sacramento, CA, 95832-1148. UNITED STATES OF AMERICA Email: [email protected]

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Appendix 2: Agenda

3RD FAO/IAEA RESEARCH CO-ORDINATION MEETING ON

"RESOLUTION OF CRYPTIC SPECIES COMPLEXES OF TEPHRITID PESTS TO OVERCOME CONSTRAINTS TO SIT APPLICATION AND INTERNATIONAL TRADE" 26 – 30 August 2013

Universidad Nacional de Tucumán, Facultad de Agronomía y Zootecnia Hotel Tucumán Center, Tucumán , ARGENTINA

MONDAY, 26 AUGUST 2013

08:30 - 08:45 Teresa Vera, Jorge Hendrichs. Opening and welcome statements. Goals of the meeting, review of agenda, administrative and other issues.

SESSION I : Chairperson: Gary Steck

08:45 - 09:15 Iara Sordi Joachim-Bravo, Raul Laumann, Vanessa Simões Dias, Alzira Kelly Passos Roriz, Clarissa Petitinga, Beatriz Jordão Paranhos, Aldo Malavasi. Analysis of sexual compatibility among Anastrepha fraterculus populations from Brazil.

09:15 - 09:45 J. Rull, S. Abraham, M. Mendoza, M.C. Liendo, F. Devescovi, D.F. Segura, K. Roriz, N. Nolasco, R. Castañeda, E. Tadeo, R. Brizova, A. Kovaleski and M.T. Vera. Evolution of reproductive isolation among different morphotypes of Anastrepha fraterculus.

09:45 - 10:15 Suksom Chinvinijkul, Sunyanee Srikachar, Weerawan Amornsak and Sujinda Thanaphum. .Field cage mating compatibility test of Bactrocera dorsalis complex of the north and the south of Thailand.

10:15 - 10:45 Ruth R. do Nascimento, Lucie Vanicková, Adriana L. Mendonça, Alana L. Mendonça, Beatriz Jordão Paranhos, Blanka Kalinová and Antônio Euzébio Goulart Sant´Ana. Chemical studies of the sex pheromone of five Brazilian populations of Anastrepha fraterculus (Diptera: Tephritidae).

10:45 - 11:00 BREAK

11:00 - 11:30 Peter Teal. Volatile chemicals released by Tephritid flies as a tool to understanding species diversity.

11:30 - 12:00 Radka Břízová, Lucie Vaníčková, Blanka Kalinová, Michal Hoskovec, Ruth Rufino Do Nascimento. Pheromones as an indispensible instruments 109

for distiction of Anastrepha fraterculus by age and geographic origin.

12:00 - 12:30 Suk-Ling Wee, Alvin Kah-Wei Hee and Keng-Hong Tan. Emission of male sex pheromone by four cryptic species, Bactrocera dorsalis, B. invadens, B. papayae and B. philippinensis, following Methyl Eugenol consumption.

12:30 - 13:00 Blanka Kalinová, Radka Břízová, Maria Faťarová, Lucie Vaníčková, Michal Hoskovec. Pheromones of three species of Ceratitis FAR complex.

13:00 - 14:00 LUNCH

SESSION II : Chairperson: Sunday Ekesi

14:00 - 14:30 Lucie Vaníčková, Massimiliano Virgilio, Aleš Tomčala, Radka Břízová, Adriana de Lima Mendonça, Blanka Kalinová, Ruth Rufino Do Nascimento, and Marc De Meyer. Cuticular hydrocarbons as a tool for resolution of the fruit fly species complexes.

14:30 - 15:00 Ishan ul Haq, Marc J.B. Vreysen, Todd E. Shelly, Jorge Hendrichs. Methyl eugenol sensitivity and its effects on intraspecific mating of three sibling species of the Bactrocera dorsalis complex.

15:00 - 15:30 Alvin Kah-Wei Hee, Suk-Ling Wee and Keng-Hong Tan. Comparative sensitivity of males of four globally important cryptic species in the Bactrocera dorsalis complex - B. dorsalis, B. invadens, B. papayae and B. philippinensis, to Methyl Eugenol and subsequent male antennal protein profiles.

15:30 - 15:45 BREAK

15:45 - 16:15 Elena Drosopoulou, A. Augustinos, A. Gariou-Papalexiou, C. Caceres- Barrios, K. Bourtzis, P. Mavragani-Tsipidou, A. Zacharopoulou. Cytogenetic studies in members of Bactrocera dorsalis species complex: A comparative analysis.

16:15 - 16:45 Nelson A. Canal, Julio C. Carranza, Daniel Zabala, María del Rosario Castañeda. Population structure of the Andean morphotype of the Anastrepha fraterculus complex.

16:45 - 17:15 María del Rosario Castañeda, Daniel Zabala, F. M. Patarroyo, Julio C. Carranza, Nelson A. Canal. Population studies of Anastrepha obliqua (MacQuart, 1835).

17:15 - 17:45 General Discussion

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TUESDAY, 27 AUGUST 2013

08:30 - 08:45 Teresa Vera, Jorge Hendrichs. Announcements, adjustments to the agenda, administrative and other issues.

SESSION III : Chairperson: Blanka Kalinova

08:45 - 09:15 Janisete G. Silva, Katia M. Lima, Norman B. Barr, Raul Ruíz-Arce, Gary J. Steck, Bruce. A. McPheron, Roberto A. Zucchi. Use of mtDNA and nuclear markers to delimit and diagnose Brazilian species within the Anastrepha fraterculus complex.

09:15 - 09:45 L. Paulin, P. Gómez Cendra, M. Juri, S. Lanzavecchia, M. T. Vera, J. Cladera, J. C. Vilardi. Landscape genetics approach to study Anastrepha fraterculus populations.

09:45 - 10:15 Karen F. Armstrong, L. M. Boykin, A. Chomic, M. K. Schutze, A. R. Clarke. Molecular genetic resolution of pest species within the Bactrocera dorsalis complex

10:15 - 10:45 Anna R Malacrida, Nidchaya Akeratawong, Silvia Lanzavecchia, Blanka Kalinova, Giuliano Gasperi. Looking for species-specific sequences in Bactrocera , Anastrepha and Ceratitis species.

10:45 - 11:00 BREAK

11:00 - 11:30 Qinge Ji. Bactrocera dorsalis complex species in mainland China.

11:30 - 12:00 Nidchaya Aketarawong, Siriwan Isasawin, Anna R. Malacrida, Giuliano Gasperi, Sujinda Thanaphum. Evaluation of genetic sexing strain, Bactrocera dorsalis Salaya1 for its species complex members and molecular characterization of Y-chromosome sequences of Bactrocera dorsalis.

12:00 - 12:30 Sunday Ekesi, Chrysantus M. Tanga, Fathiya M. Khamis and Samira A. Mohamed. Host plant relationships, temperature tolerance and mating compatibility studies in Bactrocera cucurbitae and Ceratitis rosa .

12:30 - 13:00 Maulid W. Mwatawala, Massimo Virgilio, Marc De Meyer. Comparative studies on behaviour and developmental physiology of Ceratitis rosa entities (R1 and R2) occurring in Tanzania.

13:00 - 14:00 LUNCH

SESSION IV : Chairperson: Peter Teal

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14:00 - 14:30 Vicente Hernández-Ortiz, Nelson A. Canal and Juan O. Tigrero. Morphometrics of wild populations from Ecuador and Colombia of the Anastrepha fraterculus complex: The eighth passenger.

14:30 - 15:00 Gary Steck. Anastrepha species of the Central Andes of Peru.

15:00 - 15:30 M. K. Schutze and A. R. Clarke . The taxonomic history of the Oriental and invasive fruit fly: from Musca ferruginea Fabricius to Bactrocera invadens Drew, Tsuruta & White.

15:30 - 15:45 BREAK

15:45 - 16:15 M. K. Schutze, S.L. Cameron, K. Mahmood, W. Bo, C. Cáceres, M.J.B. Vreysen, A. Pavasovic, A. R. Clarke. Reproductive compatibility, morphological and morphometric variation, and molecular genetic analyses of Bactrocera dorsalis sensu lato (Diptera: Tephritidae) from Africa, the Indian Subcontinent and East Asia.

16:15 - 16:45 J. Van Cann, M. Virgilio, K. J ordaens and M. De Meyer. The Ceratitis FAR complex: biogeography, host plant spectrum and morphometrics.

16:45 - 17:30 General Discussion and organization of three Working Groups: a) Anastrepha fraterculus complex, b) Bactrocera dorsalis complex, and c) Bactrocera cucurbitae & Ceratitis FAR complex.

19:00 - 22:00 Social Event

WEDNESDAY, 28 AUGUST 2013

SESSION V : Chairpersons Jorge Hendrichs and Group Leaders

08:30 - 10:30 Working Groups . Review of achievements based on original coordinated research plans and drafting of results and scientific/technical conclusions for the different genera, approaches and techniques. Review of research needs to be addressed and other activities to be implemented.

10:30 - 10:45 BREAK

10:30 - 13:00 Workin g Groups – continued

13:00 - 14:00 LUNCH

14:00 - 15:30 Working Groups – continued

15:30 - 15:45 BREAK

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15:45 - 17:30 Working Groups Presentations – Review of achievements, conclusions and recommendations on research needs and other activities for the different genera, approaches and techniques.

THURSDAY, 29 AUGUST 2013

SESSION VI : Chairpersons Jorge Hendrichs and Group Leaders

08:30 - 10:30 Working Groups – Drafting of individual research proposals for the different genera and techniques, taking into account the proposed R&D of each research team.

10:30 - 10:45 BREAK

10:45 - 13:00 Working Groups – continued

13:00 - 14:00 LUNCH

14:00 - 15:30 Working Group Presentations – Review of recommendations for the individual research proposals for the different applications of each research team.

15:30 - 15:45 BREAK

15:45 - 17:00 General Discussion – Organization of the preparation of final CRP proceedings in a special issue and potential journals to be contacted.

FRIDAY, 30 AUGUST 2013

SESSION VII : Chairpersons Jorge Hendrichs and Group Leaders

08:30 - 10:30 Working Groups – Drafting and compiling of RCM report.

10:30 - 10:45 BREAK

10:45 - 13:00 Working Group – continued

13:00 - 14:00 LUNCH

14:00 - 16:30 Working Groups – General Discussion : Agreement on content of RCM report, on information exchange mechanisms, on location of Final RCM and closure of the RCM.

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Appendix 3: Abstracts 3rd RCM Tucuman

ABSTRACTS 3 RD RCM TUCUMAN

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Evaluation of genetic sexing strain, Bactrocera dorsalis Salaya1 for its species complex members and molecular characterization of Y-chromosome sequences of Bactrocera dorsalis Nidchaya Aketarawong 1, Siriwan Isasawin 1, Anna R. Malacrida 2, Giuliano Gasperi 2, Sujinda Thanaphum 1 1Department of Biotechnology, Faculty of Science, Mahidol University, THAILAND 2Department of Biology and Biotechnology, University of Pavia, ITALY

Abstract: Bactrocera dorsalis strain Salaya 1 is a translocation-based genetic sexing character based on a brown-white pupal color dimorphism. This strain showed successful mating competitiveness with wild B. dorsalis s.s . under semi-natural condition (Fried competitiveness value (C) = 1.33). A mass release of sterile Salaya 1 males in the field resulted in suppression of an indigenous pest population (from 23 flies/trap/day to less than 1 fly/trap/day). Consequently, fruit damage was reduced from 30% to 5%. According to results, the Salaya 1 strain has been applied to wild B. carambolae , a member of B. dorsalis species complex, in order to test mating competitiveness under the semi-natural condition. However, we found that C-value was low, suggesting that incompatible mating occurs between the genetic sexing Salaya 1 strain and wild B. carambolae . The new genetic sexing strain is therefore being further developed for pest management using the sterile insect technique. In the meantime, we have a collaborative research project between University of Pavia and Mahidol University. This project aims to study Y-chromosome sequences of B. dorsalis s.s . We focus on the Y-chromosome because it plays a key role in determining the development of male individuals and may contain genes involving male fertility. Five DNA fragments were isolated from the genome of B. dorsalis s.s . (Seiberdorf strain). After amplification of several B. dorsalis s.s . strains, we found that four fragments may be a male-specific sequence and another one may be a male-enriched sequence. More experiments such as in situ hybridization will be performed to identify the location of those sequences.

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Molecular genetic resolution of pest species within the Bactrocera dorsalis complex Karen F. Armstrong 1, L. M. Boykin 1, A. Chomic 1, M. K. Schutze 2, A. R. Clarke 2

1Lincoln University, NEW ZEALAND 2School of Earth, Environmental and Biological Sciences, Queensland University of Technology, G.P.O. Box 2434, Brisbane 4000, Queensland, AUSTRALIA

Abstract: As part of a comprehensive genetic analysis a comparative transcriptome approach was taken to identify genetic differences amongst the pest taxa B. dorsalis ss, B. papayae, B. philippinensis, B. invadens and B. carambolae. The aim was to identify novel, potentially discriminatory gene regions based on the presence of group-specific SNPs (single nucleotide polymorphisms) which might support the species status of these groups or otherwise. After filtering out non-insect EST’s (9% other organisms; 35% not annotated) ~37,000 EST’s were screened for private SNPs, each of which then manually checked for authenticity. Of many candidate markers identified 22 primer pairs have been designed and further explored to date; B. occipitalis was included as a “good species” out group. Ten loci produced useful data. These together with data from three of the previous loci using the same specimens, excluding B. invadens, were examined for character-based (private SNPs) and phylogenetic- based evidence of differences between groups. Diagnostic differences were observed in several loci for B. carambolae (11 loci), B. occipitalis (8 loci), B. philippinensis (5 loci) and B. dorsalis/B. papayae (5 loci). No loci suggested differences between B. dorsalis and B. papayae. Clusters in maximum likelihood analysis reflected these separations, although the inter-group relationships were not consistent between loci. Species tree analysis has yet to be completed. In conclusion, while a species tree may clarify these relationships as might the inclusion of further loci, based on the data to date used in isolation in the context of a phylogenetic species concept, there is still no evidence to suggest that B. dorsalis and B. papayae and possibly B. philippinensis are discrete taxa. Bactrocera philippinensis does appear to have some characters that differentiate it from the others. However, this information on its own, with such small genetic distances and sampling from a narrow geographic base, would be hard to interpret as anything more than a population variant emphasised by geographic distance from the interbreeding populations in mainland Asia. 116

Pheromones as an indispensible instruments for distiction of Anastrepha fraterculus by age and geographic origin

Radka Břízová 1,2* , Lucie Vaníčková 3, Blanka Kalinová 1, Michal Hoskovec 1, Ruth Rufino Do Nascimento 3

1Institute of Organic Chemistry and Biochemistry of the Czech Academy of Science, Prague, CZECH REPUBLIC. . 2Institute of Chemical Technology Prague, CZECH REPUBLIC 3Universidade Federal de Alagoas, Maceio, BRAZIL

Abstract:

Males of Anastrepha fraterculus form leks and release sex pheromone to attract females during the reproductive behavior. 20 substances were identified in mature male emanations. Of these substances, 6 compounds were antennally active. In young males, the active compounds are present only in trace quantities. Their concentration increases with age and reaches a maximum at the time of sexual maturity. It is very likely that only these substances are part of the male sex pheromone signal.

In order to estimate whether the variability in pheromone communication correlates with reproductive isolation observed between some A. fraterculus populations, the emanations of mature males of “Piracicaba”, “Tucuman”, “Vacaria” and “Peru” populations were compared. Semi-quantitative analyses showed both qualitative and qualitative differences in signal quality. The biggest difference was observed in the “Peru” population.

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Population structure of the Andean morphotype of the Anastrepha fraterculus complex

Nelson A. Canal, Julio C. Carranza, Daniel Zabala, María del Rosario Castañeda

Universidad del Tolima, COLOMBIA

Abstract:

The South American Fruit Fly is an important pest of fruits in Latin America, however, seven morphotypes have been found in the nominal species Anastrepha fraterculus . Specimens from Colombia belong to the Andean morphotype, together with specimens from the Venezuelan highlands. Data on morphology, biology, ecology or genetics of this biological entity are lacking.

Specimens from nine localities in Colombia were collected, from two different hosts and a wide range of altitudinal distribution of the species. The main objective was to study the morphological, cytogenetic and genetic structure of the population of the biological entity through its geographical distribution. There were not statistical differences among populations based on adult morphometry, however, the range of the measurements of the taxonomic characters are wider than previously reported. The karyotype of the species was described; it differs from the other known karyotypes of the complex and it is variable among Colombian populations.

Studies of the mitochondrial genes COI and COII show eight populations in a clade differing from other populations from Latin America; the ninth population, from the South of the country, near Ecuador, was grouped apart from the other eight and it appear to be related to Ecuadorian populations, however, its karyotype and morphometry are related to other populations from Colombia and not from Ecuador.

For quarantine purposes, A. fraterculus from Colombia belongs to a single species along the Highland Mountains, from the South-West to North- East and the specimens from the South have to be studied more deeply. Aspects of biology, ecology, quarantine status, chemical ecology, among other aspects, have to be studied for this widely unknown morphotype.

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Population studies of Anastrepha obliqua (MacQuart, 1835) María del Rosario Castañeda, Daniel Zabala, F. M. Patarroyo, Julio C. Carranza, Nelson A. Canal University of Tolima, COLOMBIA Abstract: The West Indian Fruit Fly Anastrepha obliqua is distributed from Mexico to South Peru and North-East Brazil, and it is an important pest of mango and other fruits, with quarantine restrictions. The species is close to A. fraterculus and like this last species, and due to a wide distribution, some studies have shown population variability and it has been suggested that a complex of cryptic species may be present in the biological entities. It is important to clear this fact to support future SIT activities and/or quarantines at international markets.

Morphological, cytogenetic and genetic studies were performed, based on Colombian populations and including specimens from Brazil and Mexico or data from previous studies. Genetic variability was found, supported by 100 bootstrap. Cytogenetic variability was found especially in the size of sexual chromosomes and shape of C-band. Morphometric analyses show some divergent populations. Results obtained show population variability; however results are not clear at this moment because differences found are not as remarkable as the variability found within A. fraterculus .

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Field cage mating compatibility test of Bactrocera dorsalis complex of the north and the south of Thailand Suksom Chinvinijkul 1, Sunyanee Srikachar 2, Weerawan Amornsak 3, Sujinda Thanaphum 4 1Department of Agricultural Extension, Ministry of Agriculture and Cooperatives, Chatuchak, Bangkok 10900, THAILAND 2Department of Agriculture, Ministry of Agriculture and Cooperatives, Chatuchak, Bangkok 10900, THAILAND 3Department of Entomology, Faculty of Agriculture, Kasetsart University, Bangkhen Campus, Chatuchak, Bangkok 10900, THAILAND 4Department of Biotechnology, Faculty of Science, Mahidol University, Salaya Campus, Salaya, Nakhon Pathom, THAILAND

Abstract: The sexual compatibility among Bactrocera dorsalis complex (Diptera: Tephritidae) populations from the northern and southern extremes of geographical distribution of Thailand was assessed in field cages. Six wild colonies of Bactrocera dorsalis, Bactrocera papayae and Bactrocera carambolae from Chiang Mai (north) and Nakhon Si Thammarat (south) were collected from infested fruits and maintained at the fruit fly mass rearing facility in Pathumthani province. Identified flies of different populations were genetically studied, including a pheromone analysis. Intra– specific and inter-specific mating compatibility of the 2 nd - 4th generation flies was investigated at the intra-regional and inter-regional level. Bactrocera dorsalis , B. papayae and B. carambolae males 23, 28 and 28 day old and females 21, 28 and 28 day old, respectively, were used in these studies. Twenty individuals of each sex of two different colour marked populations were released into the field cages. A potted mango tree approximately 180 cm height in each octagonal field cage was set up for the experiment. Five mated pairs in each replicate were randomized and individually assessed for fecundity. Sexual compatible mating was quantified with the following indices: the Relative Isolation Index (RII), Isolation Index (ISI), Male Relative Performance Index (MRPI), and Female Relative Performance Index (FRPI). Results of the intra-species mating compatibility tests showed that B. dorsalis from the north and the south preferred to mate within region (RII=1.73), higher potential to mate appeared in males from the north than the south (MRPI=0.09), but females from the south showed higher mating (FRPI=-0.15). Bactrocera papayae showed inter-regional mating compatibility between north and south (RII=1.04), with males from the south and females from the north showing higher propensity to mate (MRPI=-0.13, FRPI=0.03). Bactrocera carambolae showed the same trend in mating as B. dorsalis that preferred to mate within region (RII=2.41), with both males and females from the south showing higher potential to mate (MRPI=-0.22, FRPI=-0.27). The inter-species mating tests between B. dorsalis and B. papayae from the north showed non-selective mating (RII= 0.98), with both males and females of B. dorsalis from the north showing higher potential (MRPI=0.19, FRPI=0.38), while in the south (RII=1.94) both males and females of B. papayae showed higher potential in mating (MRPI=-0.51, FRPI=-0.48). Inter-regional mating between B. dorsalis and B. papayae from the south showed selectivity by both males and females of B. dorsalis (RII=2.50, MRPI=0.58, FRPI=0.66). Bactrocera dorsalis showed high potential species selectivity in the north with B. carambolae (RII=2.04, MRPI=0.18, FRPI=0.32) and high inter-regional selectivity by B. dorsalis from the south (RII=5.50, MRPI=0.48, FRPI=0.90). 120

Bactrocera papayae and B. carambolae showed non-selectivity in the north (RII=0.77), with males of B. carambolae and females of B. papayae showing higher potential (MRPI=-0.37, FRPI=0.55), but selectivity in the south with males of B. papayae and females of B. carambolae showing higher potential (RII=1.92, MRPI=0.28, FRPI=-0.17). Meanwhile infested star apple fruits from Chantaburi province (east), which was recorded as the host and place that B. carambolae was presented in Thailand, were collected for reference.

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The Ceratitis FAR complex: biogeography, host plant spectrum and morphometrics

J. Van Cann 1, M. Virgilio 2, H. Delatte 3, K. Jordaens 1, 2 , M. De Meyer 2 1University of Antwerp, Antwerp, BELGIUM 2Royal Museum for Central Africa, Tervuren, BELGIUM 3CIRAD, UMR PVBMT, St Pierre, La Réunion, FRANCE

Abstract : Earlier studies on morphological and genetic differentiation of the FAR complex are shortly reviewed. They revealed the existence of five genotypes within the Ceratitis FAR complex, comprising two groupings within Ceratitis rosa (‘cold’ and ‘hot’), two within Ceratitis fasciventris (‘dark’ and ‘pale’) and Ceratitis anonae . These different groupings were confirmed by morphological differences in the male secondary characters, in particular the leg ornamentation.

Distributional data for C. rosa and C. fasciventris were re-examined. For C. fasciventris the two types are largely separated in a West-Central group (‘dark’ type) and an eastern group (‘pale’). However, isolated occurrences of the ‘dark’ type are found in Angola, Malawi and Tanzania. For C. rosa the two types seem to be separated along temperature clines with the ‘cold’ type restricted to lower latitudes (most part of South Africa) and higher altitudes. For the ‘cold’ type, precipitation also seems to have an impact on the distribution.

Host plant information is inconclusive. All types are polyphagous with a large overlap of host plants, especially regarding those of economic significance. In C. rosa there is a tendency for the ‘cold’ type to be found more in fruits grown in temperate climates, compared to the ‘hot’ type.

Morphometric studies, based on wing landmarking, indicate a clear sexual dimorphism. There is also an indication of separation between the three established taxa ( C. fasciventris , C. anonae , C. rosa ) and to a lesser extent, between the five genotypes.

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Chemical studies of the sex pheromone of five Brazilian populations of Anastrepha fraterculus (Diptera: Tephritidae) Ruth R. do Nascimento 1, Lucie Vanicková 1,2 , Adriana L. Mendonça 1, Alana L. Mendonça 1, Beatriz Jordão Paranhos 3, Blanka Kalinová 2, Antônio Euzébio Goulart Sant´Ana 1 1Universidade Federal de Alagoas, BRAZIL 2Institute of Organic Chemistry and Bichemistry of the Czech Academy of Prague, CZECH REPUBLIC 3Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA), BRAZIL

Abstract: The sex pheromone mixtures released by five Brazilian laboratory populations of A. fraterculus from two different geographical regions: four from the South and one from the Southwest of Brazil were studied and the obtained results were compared with those found for the populations collected in the estate of Maceió, Alagoas (Northeast of Brazil) and Tucumán, Argentina. Prior chemical studies, the populations were established under laboratory conditions and as soon as the males reached sexual maturity, experiments aiming to collect the volatiles released by calling males of all populations were performed in order to obtain extracts containing the volatile molecules used by calling males as sex pheromone. These extracts were analyzed by GC x GC/TOF-MS to determine its chemical composition as well to quantify the amount of each compound within the volatile mixtures. These analyses showed that 14 compounds were common to all studied populations, but their relative proportions vary among the populations. It was also observed that six out of the ten compounds found to elicit electrophysiological response on A. fraterculus females from a laboratory population derived from Tucumán, Argentina and maintained in Seibersdorf, Austria were also present in the volatile mixtures of compounds released by males of the studied Brazilian populations. From the obtained results we can reach the following conclusions: (i) Significant quantitative differences were found in the volatile mixtures released by A. fraterculus males from the five Brazilian populations studied; (ii) The mixtures of compounds from all studied populations are similar, but they do differ in the proportions of components; (iii) The five populations form five distinct groups, but a degree of overlapping is clearly observed among two populations from the south of Brazil: Bento Gonçalves and São Joaquim and one from the Southeast: Piracicaba, and (iv) The populations from Vacaria, Pelotas and Alagoas form three distinct groups. 123

Cytogenetic studies in members of Bactrocera dorsalis species complex: A comparative analysis Elena Drosopoulou 1, A. Augustinos 2,3,4 , A. Gariou-Papalexiou 2, C. Cáceres-Barrios 4, K. Bourtzis 4, P. Mavragani-Tsipidou 1, A. Zacharopoulou 2 1Department of Genetics, Development and Molecular Biology, School of Biology, Faculty of Sciences, Aristotle University of Thessaloniki, GREECE 2Department of Biology, University of Patras, GREECE 3Department of Environmental and Natural Resources, University of Patras, Agrinio, GREECE 4Insect Pest Control Laboratory, Joint FAO/IAEA Programme of Nuclear Techniques in Food and Agriculture, Vienna, AUSTRIA Abstract: In the previous meeting we reported on the polytene chromosome maps of the five members of the B. dorsalis complex under study ( dorsalis s.s., philippinensis, papayae, carambolae and invadens ), pointing to the non-detection of chromosomal rearrangements (mainly inversions) that could act as diagnostic characters and as barriers to gene flow among the different members of the complex. However, the above analysis cannot rule out the presence of small rearrangements, undetectable by microscopic observation. In respect to this, we focused on two directions: a) the cytogenetic analysis of bidirectional F 1 hybrids of B. dorsalis s.s with B. invadens and B. carambolae, and b) the in situ hybridization of several DNA markers including unique genes on the polytene chromosomes of different members of the complex, as well as of the aforementioned hybrids. Cytogenetic analysis and in situ localization of unique genes in the hybrids could reveal the presence of smaller rearrangements among the parental taxa. Although ongoing, our main findings can be summarized as follows: a) no extended asynapses or rearrangements were observed in the two F 1 hybrids, b) a specific inversion was observed in both hybrids always as heterozygous, which derives from one of the polymorphic inversions observed in the B. dorsalis s.s genome, c) there seem to be some minor asynapses especially in the B. dorsalis s.s X B. carambolae F1 hybrids, probably attributed to differential expression (through differential puffing activity) of the parental chromosomes, and d) preliminary in situ hybridization results of six unique genes give no evidence of small chromosomal rearrangements and verify the accuracy of the proposed polytene chromosome maps of the dorsalis complex as well as the proposed homologies with other Tephritid polytene chromosome maps. To summarize, up to now we have no evidence of chromosomal differences that could have enhanced the limitation of gene flow among the different entities of the complex. Therefore, it is unclear how these entities could have evolved and maintained as different species, especially in sympatry. The existence of workable polytene chromosome maps, along with the localization of more unique genes and markers derived from genome sequencing and EST projects of B. dorsalis s.s. could facilitate direct synteny comparisons among the members of the complex. In addition, they could support the finishing of ongoing genome projects, which in turn can shed light to the actual species boundaries in the complex.

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Host plant relationships, temperature tolerance and mating compatibility studies in Bactrocera cucurbitae and Ceratitis rosa Sunday Ekesi, Chrysantus M. Tanga, Fathiya M. Khamis, Samira A. Mohamed

International Centre of Insect Physiology & Ecology (ICIPE), Nairobi, KENYA

Abstract:

During the period under review, the following program of work was carried out (1) establish lowland and highland populations of Ceratitis rosa and study their temperature tolerance, assess mating compatibility between the two populations and share materials with CRP participants; and (2) establish cultures of Bactrocera cucurbitae from different hosts and habitats, catalogue their host plants and carry out host preference studies, preferably under field cage conditions. We were able to successfully rear the lowland population of Ceratitis rosa from guava fruits collected from Mwanjamba, Msambweni, Kenya, while the highland populations was reared from mango fruits collected from Kithoka, Meru, Kenya.

Across generations, larval duration of the highland population was longer (20.1-22.4 d) when compared with the lowland population (16.2-18.7 d). Similarly, across generations, percentage pupal recovery of the highland population was higher (30.5 to 66.4%) compared to the lowland population (15.8 to 42.6%). In both populations, pupal recovery increased from parent generation to the fourth generation. Pupal weight was not affected by population or generation, but became heavier with increasing generations. Significantly more adults emerged from the highland population (70.6 to 78.2%) compared with the lowland population (46.2 to 56.2%). Ten day fecundity was not affected by population, but increased with increasing generations of rearing. Across generations, egg fertility in the highland population was significantly higher (50.4 to 70.2%) than that of the lowland population (34.5 to 48.9%). Temperature had a significant effect on development and survival of both populations of C. rosa .

Egg development for highland population ranged from 1.8 d at 33 0C to 7.8 d at 15 0C, while that of the lowland population ranged from 1.6 d at 33 0C to 7.5 d at 15 0C. At the larval stage, development ranged from 9.5 d at 33 0C to 24.9 d at 15 0C in the highland population while in the lowland population, it took 7.7 d at 35 0C to 22.9 d at 15 0C. Pupal development ranged from 10.9 d at 30 0C to 35.4 d at 15 0C in the highland population. The developmental time of the pupal stage from the lowland population ranged from 9.1 d at 30 0C to 34.3 d at 15 0C. Regression analysis showed a strong positive linear relationship between temperature and developmental rate for all stages. Lower developmental thresholds for all stages were estimated and will be presented. Parameter estimates for the Brière-1 nonlinear model predicted the lower and upper temperature thresholds for all stages. At the egg stage, survival rates ranged between 43.5% at 33 0C to 63.5% at 25 0C in the highland population and 72.0% at 35 0C to 91.5% at 25 0C in the lowland population. In the highland population, survivorship at the larval stage ranged between 35.8% at 33 0C to 77.0 % at 25 0C while in the lowland population survival rate ranged between 48.25% at 35 0C to 89.25% at 250C. At the pupal stage, survival rate ranged from 61.3% at 30 0C to 94.0% at 25 0C in the highland population. In the lowland population, survival rate ranged from 75.5% at 30 0C to 95.5% at 25 0C. Results obtained from mating compatibility studies showed a high degree of mating incompatibility between the 2 populations of C. rosa and also between the 2 populations and C. fasciventris . The ISI values ranged from 0.75 to 0.90 with the highland C. rosa x the lowland C. rosa showing the highest degree of isolation. All matings were achieved by males 125 of the lowland C. rosa population. No significant difference was observed in latency to mate among the mating combinations. Mating duration was also similar for all mating combinations. Generally, over 80% of the mating occurred on the tree canopy for all the combinations. Overall, this preliminary observation provides some indication of the existence of variability among populations of C. rosa in Kenya. In the studies involving the host range of B. cucurbitae , the insect was reared from a total collection of 17 plant species comprising 10 families covering Cucurbitaceae, Solanaceae, Anarcadiaceae, Rutaceae and Myrtaceae from surveys carried out at the Coast, Eastern and Central Provinces of Kenya. Across the localities, highest infestation was recorded from Momordica charantia (9.8 to 16.2 flies/kg fruits), Citrullus lanatus (8.5 to 11.2 flies/kg fruits) and Lycopersicum esculentum (5.7 to 12.1 flies/kg fruits). The next most important group of fruits included Cucumis sativus , Cucurbita maxima and C. moschaata with infestation indices ranging from 2.6 to 4.4 flies/kg fruits. In general, there was a tendency for higher fruit infestation in Taveta (lowland) than in Muranga and Nthagaiya (highland). Bactrocera cucurbitae was also recorded from non-traditional host plants such as Mangifera indica, Citrus sinensis, C. reticulata and Psidium guajava, but infestation did not exceed 0.5 flies/kg fruits. In some fruit species, B. cucurbitae frequently shared host with B. invadens and a variety of Dacus spp. Future research activities should also address competitive interactions among the different fruit fly species especially on cucurbits. Colonies of two populations of B. cucurbitae (from tomato and bitter gourd) have been established using the whole fruit rearing technique. In host preference studies conducted in field cages, results showed that significantly more puparia were recovered from M. charantia (134.2) compared to the other host fruit species in experiments in which bitter gourd populations of B. cucurbitae were used. The next preferred lots of fruits were C. lanatus, C. sativus, C. maxima and C. mosschata with infestation ranging from 74.3-76.5. In experiment involving the tomato population of B. cucurbitae , the insect exclusively preferred tomato (117.2 puparia) compared to the other host plants (10.6-18.4 puparia). In this particular case, no puparia were recovered from guava and mango. In both populations of B. cucurbitae and on all host plants, adult emergence ranged from 78-82% and did not differ significantly across treatments. We conclude that populations of B. cucurbitae vary in their preference to host plants and this preference is driven also by variation among population of the pest. During the period under review, fruit fly specimens (specifically C. rosa ) of different developmental stages and quantities were shipped to Dr. Gary Steck (USA), Dr. Marc De Meyer (Belgium) and Dr. Lucie Vanickova (Czech Republic) for different activities related to the CRP.

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Methyl eugenol sensitivity and its effects on intraspecific mating of three sibling species of the Bactrocera dorsalis complex Ihsan ul Haq 1,2 *, Marc J.B. Vreysen 1, Todd E. Shelly 3, Jorge Hendrichs 4 1Insect Pest Control Laboratory, Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, Seibersdorf, AUSTRIA 2National Agricultural Research Centre, Park Road, Islamabad, PAKISTAN 3USDA-APHIS, 41-650 Ahiki Street, Waimanalo, HI 9795, USA 4Insect Pest Control Section, Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture Vienna, AUSTRIA

Abstract: Bactrocera dorsalis Hendel, Bactrocera carambolae Drew & Hancock, and Bactrocera invadens Drew, Tsuruta & White are included in the Bactrocera dorsalis species complex (Diptera: Tephritidae). They are methy eugenol (ME) responsive and the effects of ME in enhancing the male mating success are uniform across the B. dorsalis species complex, but the sensitivity of males within the complex differs.

Effects of ME (feeding or no feeding to either or both types of males) on mating compatibility between B. dorsalis and B. carambolae , and B. dorsalis and B. invadens were studied. The sensitivity of all three males to different doses of ME (0.05 ml, 0.1 ml, 0.5 ml) was also measured by recording the numbers of males arriving at a ME source over the duration of 1 h. All experiments were run under field cage conditions.

ME had no effect on reducing the mating incompatibility between B. dorsalis and B. carambolae ; they remained relatively incompatible with each other regardless of the treatment. On the other hand B. dorsalis and B. invadens were compatible without and with ME exposure (no isolation).

ME seems to increase the total number of matings achieved when given to both types of males and when given to either B. carambolae or B. dorsalis males only. ME feeding to B. invadens males only did not increase the total number of their matings. Sensitivity to ME across the three taxa was inversely proportional to the dose of ME, indicating that further ME sensitivity testing needs to be carried out at a lower range of doses.

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Morphometrics of wild populations from Ecuador and Colombia of the Anastrepha fraterculus complex: The eighth passenger

Vicente Hernández-Ortiz 1, Nelson A. Canal 2, Juan O. Tigrero 3

1Instituto de Ecología A.C., MEXICO 2Universidad del Tolima, COLOMBIA 3Escuela Politécnica del Ejército, ECUADOR

Abstract:

In previous studies we made a comprehensive morphometric analysis including a total of 32 populations of the Anastrepha fraterculus complex, originating from Mexico to Argentina. The most outstanding achievement of this investigation was the development of linear morphometric techniques for the recognition of 7 morphotypes within the AF species complex (sensu Hernandez-Ortiz et al. 2012).

Under the approach of linear and geometric morphometrics, 10 Colombian and 7 Ecuadorian populations were examined, all coming from different locations and altitudinal strata along both countries, which were compared with other populations along the Neotropics.

Our results showed that all of the Colombian populations belong to a single taxon, and those are consistent with the Andean morphotype previously described. In contrast, populations coming from Ecuador exhibited clear differences among them, so that, preliminary interpretations suggested that they belong to two distinct morphotypes; the Peruvian morphotype distributed in the lowlands, and a new Ecuadorian morphotype which occurs in the highlands, that proved to be different from all others studied before. Therefore, these findings support the presence of the eight morphotype within the Anastrepha fraterculus complex.

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Bactrocera dorsalis complex species in mainland China Qing Ji Institute of Beneficial Insects, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, CHINA Abastract: Based on the trapping network established last year, Methyl eugenol (ME) and Cuelure (Cue) traps were still used for trapping fruit flies in mainland China. Totally more than 35,000 specimens of the Subfamily Dacinae were collected from Henan (Luoyang), Jiangsu (Jintan, Nanjing), Chongqing, Jiangxi, Fujian (Xiamen, Fuzhou, Zhangzhou, Gutian and Putian), Guangxi (Nanning, Guilin), Guangdong (Dongguan, Guangzhou), Guizhou (Guiyang), Yunnan (Jinghong, Yuanjiang and Jianshui) and Hainnan (Wenchang, Danzhou and Sanya), and then studied and identified. 17 species of Bactrocera Macquart were identified and they were B. bivittata Lin et Wang, B. philippinensis Drew et Hancock, B. correcta (Bezzi), B. tuberculata (Bezzi), B.dorsalis (Hendel), B. flavoscutellat a Lin et Wang, B. wuzhishana Lin et Yang , B. thailandica Drew et Hancock, B. rubiginus Wang et Zhao, B. latifrons (Hendel), B. cheni , sp. nov., B. guiyangensis , sp. nov., B. hardyi , sp. nov., B. jinghongensis , sp. nov., B. nanningensis , sp. nov., B. nigrifacia , sp. nov., and B. zonata (Saunders) All specimens are deposited in Fujian Agriculture and Forestry University. In the meantime, we cooperated with local counterparts to collect damaged fruits in Yunnan, Guangdong, Guizhou, Hainan, Jiangxi, Hubei, Hunan, Jiangsu, Guangxi and Fujian for rearing in the laboratory. Laboratory strains from Hangzhou, Shanghai, Hunan, Guangdong, Yunnan, Hainan, Hubei and Guangxi were established. In addition theree papers were published:

Zhang Nan-Nan, Ji Qing-E, Chen Jia-Hua. 2011. Three new species and one new record of the Genus Bactrocera Macquart (Diptera, Tephritidae) from Yunnan, China. Acta Zootaxonomica Sinica 36 (3):598-603. Zhang Nan-nan, Ji Qing-e, Yang Jian-quan, Chen Jia-hua. 2011. A new species of the Genus Bactrocera Macquart (Diptera, Tephritidae: Dacinae) from China. Entomotaxomomia 33 (4):262-266. Zhang Nan-Nan, Ji Qing-E, Chen Jia-Hua . 2012. A new species of the genus Bactrocera Macquart from China (Diptera, Tephritidae). Acta Zootaxonomica Sinica 37 (1):206-208.

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Analysis of sexual compatibility among Anastrepha fraterculus populations from Brazil

Iara Sordi Joachim-Bravo 1, Raul Laumann 2, Vanessa Simões Dias 3, Alzira Kelly Passos Roriz 3, Clarissa Petitinga 3, Beatriz Jordão Paranhos 3, Aldo Malavasi 3

1Universidade Federal da Bahia (UFBA), BRAZIL 2CENARGEN-EMBRAPA, EMBRAPA–Semiárido, BRAZIL 3Medfly Rearing Facility Brazil (Moscamed), BRAZIL

Abstract:

Anastrepha fraterculus, known as South American fruit fly, has been considered as the most important pest of native fruits in South America and it is a major pest in apple orchards in south of Brazil. The high level of variability among natural population of A. fraterculus suggests that is a complex of sibling species and not a single biological entity.

In Brazil genetic and behavioral studies on mating choice support the hypothesis of at least three different species group in the country. The definition of their taxonomic status will support the implementation of a SIT program for A. fraterculus in Brazil.

In this paper we finished the evaluation of pre-zygotic sexual compatibility between 4 southern populations, 1 southeast population and 1 northeast population of Brazil. We also evaluated some aspects of courtship behavior and acoustic communication during courtship.

The populations from southern Brazil (Vacaria, Pelotas, Bento Gonçalves and São Joaquim) showed full mating compatibility, supporting preliminary data presented in the last report. These populations seem to belong to the same biological entity.

Comparisons among southern populations (Vacaria, Bento Gonçalves and São Joaquim) and southeast population (Piracicaba) showed a greater degree of mating incompatibility, also corroborating last report’s data. Contrary to initial expectations, results of tests between a southern population (Bento Gonçalves) and a northeast population (Parnamirim) showed mating compatibility.

The analysis of comparative frequency of behavioral units of southern and southeastern populations and of Parnamirim (northeast region) showed differences concerning the behavioral units’ frequency related to copula success (Arrowhead, Attempt, Calling, Fanning, Graceful + Spin). The data showed a distinction between wild populations (Vacaria and Bento Gonçalves) and laboratory-reared populations for several years (São Joaquim and Piracicaba). Additional experiments must be done to elucidate the relevance of these data and complete the comparisons between populations.

Concerning acoustical communication, data permitted to compare pulse duration, pulse period (repetition time) and dominant frequency (or fundamental frequency) of 4 populations (São Joaquim, Vacaria, Bento Gonçalves and Piracicaba) for short pulses and of 3 populations (São Joaquim, Vacaria and Bento Gonçalves) for long pulses. In most of the cases, there was a significant variation in the acoustic parameters between individuals inside each population, but when differences in parameters of each population were tested no significance were found. Significant differences were found in dominant frequency for short pulses (population of Piracicaba show the higher frequencies in short pulses) and in pulse 130 duration and pulse period for long pulses (São Joaquim population showed the longest pulses and pulses periods). Cluster analyses did not show any specific group formation between individual of different populations suggesting that the parameters of the acoustic signals of males of A. fraterculus from different geographic populations are not involved in sexual isolation.

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Comparative sensitivity of males of four globally important cryptic species in the Bactrocera dorsalis complex - B. dorsalis, B. invadens, B. papayae and B. philippinensis, to methyl eugenol and subsequent male antennal protein profiles

Alvin Kah-Wei Hee 1, Suk-Ling Wee 2, Keng-Hong Tan 3

1Department of Biology, Faculty of Science, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, MALAYSIA

2School of Environmental & Natural Resource Sciences, Faculty of Science & Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor Darul Ehsan, MALAYSIA

3Phi-Biotech Sdn. Bhd., 20, Jalan Tan Jit Seng, 11200 Tanjong Bungah, Penang, MALAYSIA

Abstract:

Males of certain putative species belonging to the Bactrocera dorsalis complex are strongly attracted to, and readily feed on methyl eugenol that is found in over 480 plant species worldwide. Amongst those species in the complex are some of the world’s most severe fruit pests such as the Oriental fruit fly, B. dorsalis, along with its very close sibling members, B. papayae , B. philippinensis and B. invadens . Whilst the first three species continue to threaten the fruit industry in the Asia-Pacific countries, the rapid establishment of B. invadens in Africa within the last decade had raised an alarm on this pest that can potentially devastate the entire fruit industry there.

The identification of these four cryptic species has been the subject of great controversy for a number of years due to their very similar morphology, mating compatibility, production of viable offspring and sex pheromone system following pharmacophagy of methyl eugenol, an extremely potent male fly attractant.

In the present study, the comparative sensitivity of B. dorsalis s.s. , B. papayae , B. philippinensis and B. invadens to methyl eugenol is being investigated. To date, laboratory trials have demonstrated that the ED 50 (effective median dose required to elicit 50% of male fruit flies tested) of the four species showed no significant difference.

In addition, gel electrophoretic analyses of the male antennal proteins extracted from the four male sibling species following exposure to methyl eugenol did not show differences. However, efforts are currently made to evaluate all the male antennal proteins on a finer scale using the high performance microfluidic capillary electrophoresis. Results of this work as well as the current behavioural response of the males to methyl eugenol will be discussed in the meeting.

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Pheromones of three species of Ceratitis FAR complex

Blanka Kalinová 1, Radka Břízová 1,2 , Maria Faťarová 2, Lucie Vaníčková 3, Michal Hoskovec 1

1Institute of Organic Chemistry and Biochemistry of the Czech Academy of Science, Prague, CZECH REPUBLIC 2Institute of Chemical Technology Prague, CZECH REPUBLIC 3Universidade Federal de Alagoas, Maceio, BRAZIL

Abstract:

Fruit fly males of many species form leks and release sex pheromone to attract females during the reproductive behavior. Male sex pheromones are highly species-specific and can therefore help in taxonomic categorization or in species recognition.

Previous research has demonstrated the multi-component nature of the pheromones and showed that Ceratitis rosa and Ceratitis anonae share some pheromone components, but are also characterized by certain species-specific substances.

At the upcoming meeting we will report on the composition of the male pheromone in Ceratitis fasciventris. Like in C. rosa and C. anonae, male sex pheromone in C. fasciventris is multi-component with shared and specific substances .

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Looking for species-specific sequences in Bactrocera, Anastrepha and Ceratitis species Anna R Malacrida 1* , Nidchaya Akeratawong 2, Silvia Lanzavecchia 3, Blanka Kalinova 4, Giuliano Gasperi 1

1Department of Biology and Biotechnology, University of Pavia, 27100 Pavia, ITALY 2Department of Biotechnology, Mahidol University, Bangkok 10400, THAILAND 3Instituto de Genética “Ewald A. Favret” (INTA), B1712WAA Castelar, Buenos Aires, ARGENTINA 4Institute of Organic Chemistry and Biochemistry ASCR, CZ-166 10 Prague 6, CZECH REPUBLIC *The Pavia laboratory actively contributed to this work: Ludvik M. Gomulski, Francesca Scolari, Marco Falchetto, Mosè Manni.

Abstract: Our aim is the identification and characterization of species and sex-specific sequences:

- to determine the intra/inter-population diversity, the species range/shift, and the indigenous or invasive status of newly observed outbreaks.

- to provide insides into genes/expressions that could be associated with barriers to gene flow.

Our attention has been addressed to Bactrocera dorsalis ss , to Anastrepha fraterculus and to Ceratitis capitata :

a) for B dorsalis ss we have developed an integrated database of SSR genotypes from populations across the species range for traceability assays. Based on genetic data we have tried to disentangle the possible major factors that affected the invasion processes. We have identified sequences that allow to mark the Y chromosome;

b) for Anastrepha fraterculus we have developed SSR markers which allow to assess the intra/inter-population and strain differentiation, and the mating/reproductive behaviour; c) for Ceratitis capitata , using genomic/functional genomic approaches, we have identified sequences correlated to the reproduction and chemoreception that may explain some traits of its behaviour and provide species-specific markers.

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Comparative studies on behaviour and developmental physiology of Ceratitis rosa entities (R1 and R2) occurring in Tanzania

Maulid W. Mwatawala 1, M. Virgilio 2, M. De Meyer 2

1Sokoine University of Agriculture, TANZANIA 2Royal Museum for Central Africa, BELGIUM

Abstract:

Determining spatial distribution of C. rosa entities along an altitudinal transect Studies were conducted along an altitudinal transect in the Morogoro Region of Tanzania. The transect lies in an embranchment of the Uluguru mountains, which is part of the Eastern Arc Mountains. The vegetation consists of cultivated land (maize, sugar cane, beans) fruit orchards (mango, citrus, peach, apple, jambolan, avocado, papaya, feijoa, guava) and fallow land overgrown with grasses and shrubs. Peach, apple and pear are temperate fruits and according to their climate demands they can be grown at high altitude. Most tropical fruits do not occur above a certain altitudinal limit for the same reason. The crops are cultivated by terracing and are grown in polycultures.

Modified McPhail® traps (Scentry Cie, Bilings, MT, VS) were set at ten stations along the transect (500-1650 m) approximately at 100 m altitudinal intervals. Three sets of traps, each baited with either trimedlure, EGO lure or terpinyl acetate were set on the same tree, replicated three times at each station. Traps were serviced every two weeks, for three months. The captured flies were brought to the lab where the specimens were counted and identified and finally preserved in 70% ethanol. At each site a datalogger (iButton® Maxim Integrated Products, Dallas) was placed to record temperature and relative humidity.

Testing mating compatibility of C. rosa entities (R1 and R2)

Establishment of fruit fly colonies

The studies are being carried out in laboratories at the Sokoine University of Agriculture (SUA). Colonies of the two C. rosa entities are being established from areas where they occur in Morogoro Tanzania. Sampling of host fruits concentrated in the low land (550 – 1000 m asl) and in high altitude areas (1650 – 1800 m asl). General methods and indices used by Cayol et al . (1999) will be used. Pupae from both strains are placed in separate ventilated Plexiglas cages (30 by 30 by 40 cm) until emergence. After emergence, females are kept in separate rooms from males to avoid contact with the male pheromone before the tests. All flies are kept in plastic containers (40 flies in each 1-liter plastic container) with water and food (protein and sugar). At least 48 h before the test, healthy flies will be selected and marked with a dot of water-based paint (Deka) on the notum. Two colors will be used (green and red) to mark alternatively R1 and R2 flies. The field cages have been made up of screen (20 by 20 mesh), cylindrical, with flat floor and ceiling (2.9 m diameter and 2.0 m high). Each cage will be supported by a PVC frame and set over a citrus tree. Four field cages will be used. To compensate for the greenhouse effect caused by the field cage, a black shading material will be placed on top of each field cage 135

(Cayol et al. 1999). Field cage testing protocols used by Cayol et al. (1999) will be generally followed.

Comparing developmental physiologies of C. rosa entities (R1 and R2)

Establishment of fruit fly colonies

The studies are conducted in laboratories at the Sokoine University of Agriculture (SUA). Colonies of the two C. rosa entities are being established from areas where they occur in Morogoro Tanzania. Sampling of host fruits concentrated in the low land (550 – 1000 m asl) and in high altitude areas (1650 – 1800 m asl). General methods and indices used by Cayol et al . (1999) are being used.

Pupae from both strains are placed in separate ventilated Plexiglas cages (30 by 30 by 40 cm) until emergence. Adults are fed simultaneously with enzymatic yeast hydrolysate (ICN Biomedical) and sucrose in a ratio of 1:3 and water on pumice granules in different plastic containers for each species. Fruit flies rearing protocols used by Vayssières et al . (2008) are being followed. The protocols to be used in the trial will first be harmonized with those used by International Centre of Insect Physiology and Ecology (ICIPE) and Citrus Research International, of South Africa.

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Evolution of reproductive isolation among different morphotypes of Anastrepha fraterculus

J. Rull 1, S. Abraham 2, M. Mendoza 2, M. C. Liendo 3, F. Devescovi 3, D. F. Segura 3, K. Roriz 4, N. Nolasco 5, R. Castañeda 6, E. Tadeo 1, R. Břízová 7, A. Kovaleski 8, M. T. Vera 2 1Instituto de Ecología, A.C., Xalapa, Veracruz, MEXICO 2Cátedra de Terapéutica Vegetal, Departamento de Sanidad Vegetal, Facultad de Agronomía y Zootecnia, UNT, Tucumán, ARGENTINA 3Instituto de Genética “E.A. Favret”, INTA Castelar, Buenos Aires, ARGENTINA 4Universidad Federal do Bahia, BRAZIL 5SENASA, PERU 6Universidad del Tolima, Ibagué, COLOMBIA 7Infochemical Group Institute of Organic Chemistry and Biochemistry, CZECH REPUBLIC 8Embrapa Uva e Vinho, Estação Experimental de Vacaria, BRAZIL

Abstract: During the course of the previous RCM meeting we reported results of prezygotic and postzygotic reproductive isolation among three populations of the Brazil 1 morphotype (after Hernández-Ortíz et al. 2012). Experimental results and a review of biological evidence revealed that populations from Argentina and Southern Brazil belong to a single biological entity and are fully compatible (Rull et al. 2012). By contrast, adults from the Mexican morphotype exhibited strong prezygotic isolation in the form of assortative mating (vs. Brazil 1 morphotype adults), timing of sexual activity (vs. Peruvian morphotype adults), and resistance to penetration by Mexican females when mounted by heterotypic males of several origins. Hybrids between Mexican adults and adults of the Brazil 1 or Peruvian morphotypes were either less viable (fertile) or sterile (Rull et al. in press). In the case of the Andean morphotype (Colombia) the strongest form of prezygotic isolation was related to marked differences in the timing of sexual activity (Colombian adults court and mate at dusk vs. early morning or mid-day for adults of other morphotypes). We discovered some degree of postzygotic isolation between adults of the Andean morphotype and adults of the Mexican, Peruvian, Brazil 1 and Brazil 2 morphotypes. Nevertheless, F 1 hybrid fertility was partially restored in all cases (Rull et al. in preparation), supporting previous hypotheses of hybridization in the fraterculus species group as putative speciation mechanism (Segura et al. 2011). In a separate experiment, we discovered that Peruvian females tend to store less sperm than Brazil 1 females irrespective of the origin of their mate and almost one half of the crosses involving Brazil 1 males and Peruvian females were unsuccessful. Brazil 1 females were more willing to remate than Peruvian females, irrespective of male morphotype, but latency to remating was not affected by male or female morphotype (Abraham et al. 2013 submitted). Collective evidence suggests that all A. fraterculus morphotypes are reproductively isolated and that prezygotic isolation is stronger than postzygotic isolation. Such finding, and the existence of numerous sister and cryptic species, suggest that recent differentiation in the fraterculus species group may have often occurred in sympatry (Coyne and Orr 1997). References Abraham S, Rull J, Mendoza M, Liendo MC, Devescovi F, Roriz AK, Kovaleski A, Segura DF & Vera MT. Differences in female remating propensity in two morphotypes of 137 the Anastrepha fraterculus (Diptera: Tephritidae) cryptic species complex. Bulletin of Entomological Research. Submitted Coyne JA & Orr HA (1997) “Patterns of speciation in Drosophila ” revisited. Evolution 51: 295-303. Hernández-Ortiz V, Bartolluci AF, Morales-Valle P, Frías D & Selivon D . (2012) Cryptic species of the Anastrepha fraterculus complex (Diptera: Tephritidae): A multivariate approach for the recognition of South American morphotypes. Annals of the Entomological Society of America 105: 305-318. Rull J, Abraham S, Kovaleski A, Segura DF, Islam A, Wornoayporn V, Dammalage T, Santo Tomas U & Vera MT (2012) Argentinean and Southern Brazilian Populations of Anastrepha fraterculus (Diptera: Tephritidae). Bulletin of Entomological Research 102: 435- 443. Rull J, Abraham S, Kovaleski A, Segura DF, Mendoza M, Liendo M & Vera MT (2013). Evolution of pre ‐zygotic and post ‐zygotic barriers to gene flow among three cryptic species within the Anastrepha fraterculus complex. Entomologia Experimentalis et Applicata. In press Rull J, Devescovi F, Abraham S, Roriz K, Nolasco N, Castañeda R, Tadeo E, Brizova, R, Cáceres C, Segura D & Vera MT. Reproductive isolation among Colombian Anastrepha fraterculus (Diptera:Tephritidae) and four morphotypes of the Anastrepha fraterculus criptic species complex. In preparation. Segura DF, Vera MT, Rull J, Wornoayporn V, Islam A & Robinson AS (2011) Assortative mating among Anastrepha fraterculus (Diptera: Tephritidae) hybrids from two distinct populations as a possible route to radiation of the fraterculus cryptic species group. Biol. J. Linn. Soc. 102: 346-354. 138

The taxonomic history of the Oriental and Invasive Fruit Fly: from Musca ferruginea Fabricius to Bactrocera invadens Drew, Tsuruta & White M. K. Schutze, A. R. Clarke School of Earth, Environmental and Biological Sciences, GPO Box 2434, Queensland University of Technology, Brisbane 4001, AUSTRALIA. Abstract: A fly similar to the widely distributed pest tephritid Bactrocera dorsalis , the Oriental Fruit Fly, was first detected in Africa in 2003. Two years later this fly was deemed new to science and given the name B. invadens , the Invasive Fruit Fly. Bactrocera invadens is considered taxonomically different from its Asian cousin due largely to its thoracic colour patterns: predominantly red-brown in B. invadens and black in B. dorsalis . However, questions are now being asked as to whether B. invadens is truly a new species, or rather represents a morphologically variable population of the Oriental Fruit Fly (as was initially suspected by local researchers). As a component of species delimitation research within the larger B. dorsalis species complex, a review of the taxonomic history of B. dorsalis sensu stricto was undertaken and findings suggest that there are sound taxonomic reasons as to why B. invadens should be regarded as a junior synonym of B. dorsalis . Specifically, morphological characteristics described for Musca/Dacus ferrugineus – a species described in 1794 by Fabricius and now a junior synonym of B. dorsalis – are largely consistent with modern-day B. invadens . The situation is not straight forward though, with a highly complex taxonomic history that has fuelled debate for almost two centuries. As a ‘scene setter’ for the week’s discussion, we detail the history of these species and reveal that the current confusion is nothing new.

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Reproductive compatibility, morphological and morphometric variation, and molecular genetic analyses of Bactrocera dorsalis sensu lato (Diptera: Tephritidae) from Africa, the Indian Subcontinent and East Asia M. K. Schutze 1,2, S.L. Cameron 2, K. Mahmood 3, W. Bo 4, C. Cáceres 4, M. J. B. Vreysen 4, A. Pavasovic 2, A. R. Clarke 1,2 1CRC for National Plant Biosecurity, L.P.O. Box 5012, Bruce, A.C.T. 2617, AUSTRALIA 2School of Earth, Environmental and Biological Sciences, Queensland University of Technology, G.P.O. Box 2434, Brisbane 4000, Queensland, AUSTRALIA 3Pakistan Museum of Natural History, Garden Avenue, Shakarparian, Islamabad, PAKISTAN 4 Insect Pest Control Laboratory, Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, International Atomic Energy Agency, Vienna, AUSTRIA

Abstract: Bactrocera dorsalis , B. papayae, B. philippinensis, B. carambolae and B. invadens are sibling pest members within the B. dorsalis species complex of tropical fruit flies. The species status of these taxa is unclear and this confounds quarantine, pest management and general research. Bactrocera dorsalis is distributed from Pakistan to the Pacific with the Thai/Malay peninsula its southern limit. Bactrocera papayae and B. carambolae occur in the south-east Asian archipelago, B. philippinensis in the Philippines, and B. invadens in Africa and Sri Lanka. Since the 2 nd RCM in Brisbane, the QUT group has focused on two main activities. The first was the completion and write-up of previously presented work on the species delimitation of the South-east Asian members of the complex; the second the initiation (and largely completion) of new work on the delimitation of B. dorsalis and B. invadens . The latter activity was undertaken jointly with the FAO/IAEA Insect Pest Control Laboratory, Seibersdorf, and Dr Khalid Mahmood of the Pakistan Natural History Museum. Our work on the South-east Asian members of the complex has now been published in a series of five papers covering a multi-gene phylogenetic analysis, a COI population study, a microsatellite study, mating behaviour, wing shape analysis and male aedeagus measurements. Our conclusions, as previously presented, are that B. papayae and B. philippinensis should be regarded junior synonyms of B. dorsalis : there is no evidence for these flies being distinct biological species. In contrast, there is sufficient evidence available to consider B. carambolae a distinct, albeit very closely related, biological species. The new work on comparing B. dorsalis and B. invadens has followed the same integrative taxonomic approach. Pre- and post-zygotic compatibility studies conducted at the FAO/IAEA Seibersdorf laboratories have shown random mating between a Kenyan population of B. invadens and B. dorsalis populations from Pakistan and China. There is also no evidence of post-zygotic incompatibility in these crosses. In contrast, attempted crosses between the Kenyan population and a population of B. kandiensis from Sri Lanka showed high levels of mating isolation and post-zygotic incompatibility. These latter results, as well as the earlier results showing non-random mating of B. carambolae , provide confidence that field cage mating trials provide accurate measures of mating isolation between very closely related member of the B. dorsalis complex. A COI haplotype network including B. dorsalis individuals from across its entire range from Pakistan to South-east Asia and B. invadens individuals from Africa and Sri Lanka showed no evidence of any haplotype divergence between B. dorsalis and B. invadens . In stark contrast, B. kandiensis shares no haplotypes with either B. dorsalis or B. invadens . A 140 multigene phylogenetic study has also been undertaken and we hope to present the analysis of the results in Argentina. Morphometric and geometric morphometric analysis has been conducted on similarly widely distributed samples of B. dorsalis and B. invadens . Attributes studied were wing shape, thoracic colour pattern, width of the post-sutural lateral vittae and length of male genitalia. Clinal and/or geographic variation in vittae width was detected; however there was no evidence of character variation being in any way consistent with purported morphological discontinuities between B. dorsalis and B. invadens . We conclude from this work that B. dorsalis is a morphologically highly variable and widely distributed species, and that ‘ B. invadens’ represents only an incursive population of this wide-ranging species. Wing shape and body colour pattern are consistent with the invasive African population of B. dorsalis having originated from the northern subcontinent, i.e. Pakistan or similar.

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Use of mtDNA and nuclear markers to delimit and diagnose Brazilian species within the Anastrepha fraterculus complex

Janisete G. Silva 1, Katia M. Lima 1, Norman B. Barr 2, Raul Ruíz-Arce 2, Gary J. Steck 3, Bruce. A. McPheron 4, Roberto A. Zucchi 5 1Universidade Estadual De Santa Cruz, Bahia, BRAZIL 2United States Department Of Agriculture, USA 3Florida Department of Agriculture and Consumer Services, USA 4Ohio State University, USA 5Universidade de Sao Paulo, BRAZIL

Abstract:

The South American fruit fly, Anastrepha fraterculus (Wiedemann), is among the most serious agricultural pests in South America. In Brazil, A. fraterculus has been reported to infest 81 host species in 20 plant families. A series of morphological and genetic studies have revealed that A. fraterculus actually comprises a complex of multiple species. The actual number of putative species within the A. fraterculus complex and their associated biogeography is yet uncertain.

Differences among cryptic species could have significant consequences for pest quarantine, management, and eradication. Therefore, it is paramount to study populations from a wide geographic range in Brazil using mtDNA and nuclear markers in order to capture genetic diversity of this complex. Analysis of con-specific variation among Anastrepha fraterculus samples and con-generic variation among fraterculus species group samples is critical to resolving evolutionary relationships of cryptic species.

The cooperative research program has obtained over 630 expertly identified Anastrepha samples representing nearly 70 species. This includes 244 A. fraterculus specimens gathered from several states in Brazil, Argentina, and Mexico plus an additional set of 175 specimens representing 20 taxa within the fraterculus species group as well as from other Anastrepha species groups. To date, DNA has been isolated from 630 adult specimens.

We sequenced a portion of the cytochrome oxidase c subunit I from approximately 90% of the samples. Genetic diversity estimates and Neighbor Joining Tree analysis of the aligned sequences suggest structuring among populations and species. A subset of the DNA samples have been screened for variation at additional mitochondrial (ND6) and nuclear ( period , CAD, ITS, 18S, TPI) loci. Based on variation within species and among species, several of these loci have also been identified as informative on a portion of collections for the study of cryptic species. Protocols for these genetic markers have been developed and are being used to generate a multi-gene sequence data set for A. fraterculus collections.

Additional accomplishments include analysis of a subset of populations using 10 microsatellite markers developed by Drs. Silvia Lanzavecchia and Anna Malacrida, who lead another group in this CRP. Results indicate genetic structuring between some populations.

Our collaborative efforts include morphometric analyses of populations of A. fraterculus from Brazil with Dr. Vicente Hernández-Ortíz and Dr. Iara Bravo, respectively, who lead two other groups in this CRP. Brazilian populations that are being genotyped using mitochondrial and nuclear markers will also be examined for morphometric differences. This is being done 142 through collaboration with taxonomic experts at Universidade de São Paulo, Brazil (Drs. Keiko Uramoto and Roberto Zucchi) and Instituto de Ecología A.C., Mexico (Dr. Vicente Hernández-Ortíz). The morphometric and genetic data sets will be available for comparison to other data sets generated for Anastrepha species.

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Anastrepha species of the Central Andes of Peru

Gary Steck 1

1Florida Department of Agriculture, Division of Plant Industry, USA

Abstract:

Recently obtained funding for a U.S. Farm Bill project entitled "Enhancement of fruit fly immature stage identification and taxonomy" has enabled the principal investigators to undertake extensive field work in South and Central America to acquire larval and adult stages of numerous Anastrepha species for morphological and molecular studies.

We made two trips to Peru (November 2012 and January-February 2013) and made collections in Departmento Cusco where the Anastrepha fauna is poorly known. Collection efforts were successful at elevations of about 3000 m (Sacred Valley), 1100m (Echarate) and 600-800m (Pilcopata). Identification is still on-going, but at least 37 species and 3,000 specimens of adult Anastrepha were collected from traps, larvae were reared to adult stage from 14 different host plants, and a total of approximately 800 larvae have been preserved. Additional collection activities are underway in Panama to extend the geographic range of Anastrepha spp. samples for taxonomic analysis.

In Departamento Cusco, Anastrepha fraterculus s.s. is absent or rare at the lower elevations, although at least one undescribed species of the fraterculus group is present; fraterculus is present, but not dominant at mid-elevations; and it seems to be the only species present at high elevations where it is a serious pest of stone fruits.

Our preliminary round of collecting yielded too few fraterculus specimens to establish a research colony for more general studies, but we have laid the groundwork for establishing a colony in the near future - hopefully in Seibersdorf.

Preliminary DNA sequence data indicates that A. fraterculus populations in Departamento Cusco (Andean highland and Amazonian mid-elevations) are clearly different from the Pacific coastal populations of Peru. DNA sequences from the Cusco populations, as well as those from a mid-elevation population on the eastern slopes of the Andes in Bolivia, match those of widespread fraterculus populations in Argentina and southern Brazil .

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Volatile chemicals released by Tephritid flies as a tool to understanding species diversity

Peter E. A. Teal

Chemistry Research Unit, Center for Medical, Agricultural and Veterinary Entomology, USDA-ARS, 1700 SW 23 Dr., Gainesville, FL. 32604, USA

Abstract:

It is clear that the Tephritids are a wonderfully diverse group of flies. However, as is evident from the current Coordinated Research Project many times clear species identifications are next to impossible using common systematic methods. Excellent examples of cryptic species are documented among members of the Bactrocera dorsalis complex and the Anastrepha fraterculus complex.

In collaboration with the IAEA/FAO and research groups from around the world we have analyzed volatile chemicals released by males of several groups from both complexes. Chemical data show a good correlation between the blends of chemicals males release when signaling for mates and data supporting the complexes being composed of several reproductively isolated species. Indeed, there is good evidence that pheromones released by males are a primary method of reproductive isolation for these species.

There is a not only a need to identify male produced volatiles from these cryptic species but also to determine the impact of pheromones on reproductive isolation among these groups.

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Cuticular hydrocarbons as a tool for resolution of the fruit fly species complexes

Lucie Vaníčková 1*, Massimiliano Virgilio 2, Aleš Tomčala 3, Radka Břízová 3, Adriana de Lima Mendonça 1, Blanka Kalinová 3, Ruth Rufino Do Nascimento 1, Marc De Meyer 2

1Universidade Federal de Alagoas, Maceio, BRAZIL 2Royal Museum for Central Africa, Leuvensesteenweg 13, B-3080 Tervuren, BELGIUM 3Institute of Organic Chemistry and Biochemistry of the Czech Academy of Science, Prague, CZECH REPUBLIC

Abstract:

Resolution of the species complexes in Tephritidae is a very challenging task. Recently, in fruit fly family (Diptera:Tephritidae), there have been reported several cases on species complexes including the so called Ceratitis FAR complex and Anastrepha fraterculus complex. Cuticular hydrocarbons appear to be an excellent tool for chemotaxonomical resolution of these cryptic species.

In the present study, we report the use of cuticular hydrocarbon profiles of Ceratitis anonae , C. fasciventris , C. rosa , C. capitata and A. fraterculus for distinguishing the mentioned species complexes. Quantitative as well as qualitative differences between species, populations and genders are reported. The CHC profiles consist of mixture of alkanes, internally methyl-branched alkanes, alkenes and alkadienes. The species and gender-specific cuticular hydrocarbons were identified and they can be used as chemotaxonomic markers for recognizing between the species and further between the species complexes.

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Emission of male sex pheromone by four cryptic species, Bactrocera dorsalis, B. invadens, B. papayae and B. philippinensis, following Methyl Eugenol consumption

Suk-Ling Wee 1, Alvin Kah-Wei Hee2, Keng-Hong Tan 3

1School of Environmental & Natural Resource Sciences, Faculty of Science & Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor Darul Ehsan, MALAYSIA 2Department of Biology, Faculty of Science, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, MALAYSIA 3Phi-Biotech Sdn. Bhd., 20, Jalan Tan Jit Seng, 11200 Tanjong Bungah, Penang, MALAYSIA

Abstract:

Four global cryptic pest species of the Bactrocera dorsalis complex- Bactrocera dorsalis sensu stricto , B. papayae , B. philippinensis and B. invadens, are known to be strongly attracted to, and consume methyl eugenol, a phenylpropanoid and very potent male attractant, detected in at least 480 plant species from 80 families across 46 botanical orders.

Whilst chemical and behavioural analyses have led to elucidation of the explicit and implicit roles of methyl eugenol in the fruit fly-plant interaction, evidence on the sex pheromone production by males of these cryptic species following ingestion of methyl eugenol has complemented efforts to resolve the current controversy related to the taxonomic status of the four Bactrocera species.

Following methyl eugenol feeding, males of the four cryptic species are known to produce two major phenylpropanoid compounds - (E) -coniferyl alcohol and 2-allyl-4,5-dimethoxyphenol, that are temporarily stored in the rectal gland and ultimately emitted as sex pheromone during dusk prior to mating. The pheromone emission during courtship period of B. dorsalis s.s , B. invadens, B. papayae , and B. philippinensis are being investigated using SPME-GCMS analyses and results of this work will be presented and discussed in the meeting .

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Appendix 4: Working Groups

Bactrocera dorsalis complex Anastrepha fraterculus Bactrocera cucurbitae complex Ceratitis FAR complex

M. Teresa Vera (Leader) Tony Clarke (Leader) Marc De Meyer (Leader)

Andrea Bartolucci Karen Armstrong David Haymer

Radka Brizova Nidchaya Aketarawong Helene Detalatte

Carlos Caceres Suksom Chinvinijkul Sunday Ekesi

Nelson Canal Daza Elena Drosopoulou Blanka Kalinova

Maria del Rosario Alvin Kah-Wei Hee Ana Malacrida Castañeda Mark Schutze Maulid Mwatawala Jorge Cladera Keng Hong Tan Massimiliano Virgilio Vanessa Dias Peter Teal Paola Fedyszak Kenji Tsuruta Lucia Goane Ishan ul-Haq Vicente Hernandez Ortiz Antogoni Zacharopoulo Iara Sordi Joachim Bravo

Laura Juarez

Katia Manuela de Lima

Ruth Rufino do Nascimento

Beatriz Jordao Paranhos

Kelly Passos Roriz

Juan Rull

Janisete G. Silva

Gary Steck

Lucie Vanickova 148

Appendix 5: Protocols for Collection and Shipment of Live and Dead Insects for Vouchering, Rearing, Morphology, Morphometrics, Chemical Ecology and Molecular Assays

1. Labelling 1.1. Place of origin (GPS / latitude:longitude, town, country) 1.2. Host fruit, what plant insect was collected from 1.3. Date and time of collection 1.4. Person who collected the insect

Note: strict rules of how to refer to the relationship between the fly and the plants e.g. trapped in tree, found as larvae in fruit, clearly differentiate between on fruit, in fruit, ex fruit, etc.

Consider pre-designed forms that should be attached to any specimen, forms attached as an appendix

2. Live insects 2.1. Collection 2.1.1. From wild infested fruit 2.1.1.1. Fruit type described, photo 2.1.1.2. Number of fruit needed to collect 2.1.1.3. How many trees, orchards, regions were collections made from? How wide a population should the sample be collected from? 2.1.1.4. Base number of insects required for R&D 2.1.2. From harvested fruit placed in the field 2.1.3. From non-toxic traps 2.1.4. From netting, aspiration, etc 2.1.5. From laboratory cultures 2.1.6. Shipment of wild-infested fruit from the field to local laboratory – packaging to reduce package-induced mortality, temperature management, etc 2.2. Shipment 2.2.1. Common considerations 2.2.1.1. Shipping by: 2.2.1.1.1. Hand luggage for air travel 2.2.1.1.2. Booked-on package 2.2.1.1.3. Via courier 2.2.1.1.4. Via Post 2.2.1.1.5. Personally by car 2.2.1.1.6. Packaging type, size, weight 2.2.1.2. Quarantine security – FORMS!!! 2.2.1.2.1. Packaging type, size, weight 2.2.1.2.2. Description of contents 2.2.1.2.3. Liquids present 2.2.1.2.4. Contact names, addresses, phone numbers, e-mail addresses – both sender and receiver 2.2.1.2.5. Exporting country requirements 149

2.2.1.2.6. Importing country requirements 2.2.1.2.7. Courier company requirements 2.2.1.2.8. Airline company requirements 2.2.2. As pupae 2.2.2.1. Strong packaging material 2.2.2.2. Reduce desiccation 2.2.2.3. Vibration, movement of pupae causing physical damage 2.2.2.4. Access of pupae to air 2.2.2.5. Permissible transit temperature range 2.2.2.6. Quarantine security, escape of pupae, adults 2.2.3. As eggs 2.2.3.1. Transport of liquids 2.2.3.2. Reduce leakage, maintain package integrity 2.2.3.3. Eggs in water, or in agar agar solution 2.2.4. As infested fruit 2.2.4.1. Distance to travel 2.2.4.2. Time to travel 2.2.4.3. Temperature management to reduce adult eclosion 2.3. Receipt of live insects 2.3.1. Customs clearance – FORMS!!! 2.3.2. Retrieving eggs, pupae, adults from package 2.3.3. Identification and separation of species in a mixed culture, other insects, parasitoids, etc 2.3.4. Setting up laboratory culture 2.3.5. Minimum quantity of insects required to be a valid sized culture – justification of sample size, recording

3. Dead insects 3.1. Collection 3.1.1. Wild flies reared from fruit, collected from traps, nets, etc 3.1.2. Part/s of insect required for shipment 3.1.3. Retaining remains of insect for trace-back or for study in another collaborating laboratory 3.1.4. Labelling requirements for receiving agency / laboratory, etc 3.2. Shipment 3.2.1. Shipment of insect extracts e.g. DNA extracts, pheromones 3.2.1.1. Specialist handling and shipment procedures 3.2.2. Quarantine considerations as above 3.2.3. Preserved as a dried specimen or in alcohol or alternative preservatives 3.2.3.1. Alcohol concentration 3.2.4. Shipment in alcohol and importer, airline, courier requirements 3.2.4.1. Amount of alcohol 3.2.4.2. Alcohol concentration 3.2.5. Alternatives to shipment in alcohol e.g. PEG, etc 3.3. Packaging of specimens 3.4. Receipt of specimens 3.4.1. Customs clearance

4. Post receipt handling 4.1. Quarantine security requirements of the receiving institutes 150

4.2. Quarantine security facilities at the receiving institutes 4.3. Quarantine security requirements of the importing country

5. Trapping 5.1. Type of trap, type of specimen required 5.1.1. For dry specimen 5.1.2. Trapped in water 5.1.3. Trapped in a preserving solution (e.g. PEG) 5.1.4. With or without toxin

6. Collection of rectal glands and/or volatiles from live insects – See paper by K.H. Tan and work by Peter Teal 6.1. Guarding against contamination 6.2. Storage and transport in 100% alcohol 6.3. Other issues described in the paper 6.4. Collection of pheromones (e.g. work done by Peter Teal)

7. Validation of species by recognised expert 7.1. Reference material 7.1.1. Validation before experiment start 7.1.2. Validation after experiment 7.1.3. Keep Voucher Specimens 7.1.3.1. How many to keep? 7.1.3.2. In alcohol and / or 7.1.3.3. As a dried (pinned?) specimen 7.1.3.4. Preservation and vouchering these specimens for future 7.1.4. Sharing these specimens for later molecular or morphology

8. Other issues to be addressed 8.1. Feedback on how culture is progressing, return information, courtesy call / e- mail on receipt of goods, etc 8.2. Agreement reached on terms, agreements and structures for payment requirements, if necessary, for suppliers, for analysers, etc 8.3. Consider the possibility of assessing the same individual insects for mating studies, then morphological studies, then pheromone analysis, then molecular, etc. What logistics are required if each assessment is to be done in geographically distant facilities ? 8.4. Share progeny from captured wild females for study 8.5. Detailed descriptions and documentation of collection methods, dates, times, vouchering, personnel, experimental designs, etc 8.6. Collaboration and co-operation 8.7. Potential / possibility of on-line collaboration , questions, FAQs, problems and solutions 8.8. Publication considerations, intellectual property, data sharing rights, legal requirements

9. Other reports, manuals, etc that are already published / used? E.g. ICIPE, etc. 9.1. List as many other publications that are already available.

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Appendix 6: Guidelines for Performing Mating Compatibility Field Cage Test

Note: this document is an adaptation of the guidelines presented in the FAO/IAEA/USDA Manual for Product Quality Control and Shipping Procedures for Sterile Mass-Reared Tephritid Fruit Flies (2003). The QC Manual can be downloaded from http://www- naweb.iaea.org/nafa/ipc/public/ipc-mass-reared-tephritid.html and a revised version will soon be available.

Overall Objective The overall objective of the mating compatibility field-caged test is to determine the degree of sexual compatibility between two populations/strains under semi-controlled field conditions by releasing in a confined semi-natural environment (i.e. field cages with trees inside) sexually mature fertile but yet virgin flies from these two populations to observe their mating behaviour and interactions during the time of sexual activity.

Specific Objectives 1. To determine the number of mating couples obtained in each of the possible mating combinations between the two populations. 2. To characterise male calling behaviour (time, location, aggregation in leks , number and type of males in the lek , aggressive behaviour, etc.). 3. To characterise male – female interactions once the females arrive at the lek . 4. To determine mating duration and any possible correlation with the mating combination or type of cross (homotypic vs. heterotypic). 5. To determine possible changes in female postcopulatory behaviour. 6. To determine the impact on the above mentioned variables of the origin of the other population released in the field cage (presence of plasticity in the mating behaviour). 7. Determine sperm transfer. 8. Obtain couples to perform studies of postzygotic compatibility.

Discussion Mating behaviour field cage tests aimed either to evaluate mating compatibility of male performance, are the best compromise between laboratory conditions and costly and/or impractical field observations to assess tephritid fly mating behaviour under semi-natural conditions.

Sexual compatibility is the degree to which two groups of animals tend to mate randomly without regard to their group of origin rather than mating selectively with members of their own group. For tephritid SIT programmes, the sexual compatibility between the target wild population and the candidate strain or population aimed at being transferred to mass rearing intended to be used for release should be measured before initiating any large-scale operations. In some tephritid species, sexual incompatibility could reveal the presence of sexually isolated populations and, to some further extent, the presence of cryptic species previously undetected.

The FAO/IAEA/USDA QC Manual offers a standard design for field-cage tests and it is recommend that within the CRP people follows this protocol with the particular modifications presented here. Data from these tests can be used to generate simple, reproducible, meaningful indices of sexual compatibility that can be used for making comparisons between populations. Given it was concluded in previous CRPs that the 152 nutritional status, sex ratio and density of flies in relation to available canopy surface in the field cage influences test results, efforts should be made to strictly follow the standards described thereafter in order to minimize the impact of these variables on the overall results and thus permit comparisons among different research groups.

Sources and Handling of Flies Biological material can come from already established laboratory strains or from wild populations. In both cases pupae should be placed in proper conditions to allow adult emergence. Within a few hours of adult emergence, select only flying adults and separate the sexes. Sexing has to be 100% effective. Female cages in which even one male is detected cannot be used for the tests and will have to be discarded, the same is recommended for male cages in which one female has been missplaced. Then, hold the flies according to the day of emergence in laboratory cages (screen or Plexiglas) containing water and a food until sexually mature. Age of sexual maturity will vary with species or strain so in case no data is available, preliminary tests may be required to determine the appropriate testing age. The type of food to be given should be equal to all origins and should resemble the one the flies consume in nature. Moistened dry fruits such as figs is a good worldwide available option; however a source of protein is required. Preferably, the sexes should be placed into two separate rooms at a maximum density of ±40 flies per litre volume for the case of medlfy and less for bigger flies such as some Anastrepha or Bactrocera spp.

Equipment • All necessary equipment to maintain the flies in the laboratory from emergence until release in the field cage: Plexiglas cages with “Flight Ability” devices, aspirators to sex the flies, ≈1 l containers, water and food containers, etc. • All necessary equipment to mark the flies according to the chosen technique (see below): (1) fluorescent powder; (2) water-based paint, thin soft camel hair brushes and a meshed bag; (3) cake colorants to dye the diet. • Outdoor field cage, 2 m tall by 2.9 m in diameter, set up over a plant that fills a large portion of the volume of the cage (Error! Reference source not found.). The plant should be a local host plant of the fly species to be tested (citrus, guavas, etc.). Ideally, the plant should be rooted in the ground, but potted plants may suffice if ground-rooted plants are unavailable. Artificial trees may also be an option and have the advantage that flies are not exposed to plant volatiles that alter flies behaviour. Care must be taken in setting up the cages. The available foliage must provide an abundant substrate for mating behaviour, but could be lightly pruned (if necessary) so that flies will be visible to the observer. An average of 20 medium-sized leaves should be available per fly released in the field cage. If adequate foliage and light are available within the cages and if not more than 150-200 flies are released per cage (regardless of the type of field cage test done), little if any mating activity should take place on the screen of the cage. • Plastic pill vials, scintillation vials, or similar containers of about 10-20 ml preferably clear, to collect the mating couples (depending on the amount of flies released 50 – 80 per cage). • Grease pencils and/or masking tape and pens for marking vials. • Long-wave ultra-violet lamp (should flies not be marked with paint and identifiable only by the presence of fluorescent dye) or fluorescent microscope. 153

• A minimum of 4 dental cotton wicks impregnated with water should be placed per tree as source of water for the flies. • Data recording forms with pencil. • Hydro-thermometer and luxmeter.

Procedure Before releasing the flies in the cages, it would be necessary to mark them in order to distinguish at which strain/population they belong. Three methodologies are widely used and can be applied in conjunction: (1) dyeing with fluorescent powder as in operational programmes (2) painting the flies in the thorax with water based paint, or (3) providing the flies with diets dyed with non-toxic food colouring. Each technique is applied at different times. In the case of fluorescent powder, dying occurs at the pupal stage before adult emergence with approximately 1.5 gr per kilo of pupae. In the case of painting the thorax, at least 24 to 48 hours before the test, flies are marked individually by applying a small dot of paint on the dorsal surface of the thorax. Flies can be immobilize by chilling them at approximately 5° C for a few minutes or by placing them in a bag made of mosquito net (18 mesh), placing the bag on a table, and holding the mesh down gently around each fly, one at a time. Use a thin, soft camel hairbrush to apply a small drop of paint to the fly. For the case of adding the colorant to the diet, it is advisable to do it since adult emergence. A small drop of non-toxic food colouring is added to the adult food in order to obtain the same food with different colours. Flies that ingest the food have their abdomen coloured with the provided colour. Even though marking of flies with water-based paint or cake colorants appears to have no effect on mating performance field cage test results, colours used for marking strains should be randomised among replicates and brands of paints or colorants could be evaluated ahead of time to ensure that the mark does not affect fly behaviour or survival. Immediately after marking, flies are transferred to containers suitable for releasing them into the field cages with water and food in groups of 25 – 50 flies, depending on the species, per 1 litre container. On the day of the test, males are released into a screen cage and given a period of time (e.g., 15-30 min) to disperse and establish territories. The number of flies to be released depends on the species; for the case of medfly normally 50 flies of each strain and sex are released which totals 200 flies per cage while for other species it is recommended to release 25-30 flies of each sex and origin. Time of release should precede the time of peak mating for that species. Flies should be able to fly out of the container by themselves and should not be forced to do so by shaking the container or pushing flies out. Flies that are left inside the container, dead, deformed, or apparently incapable of flying should be replaced. Then, females are released. Starting time of mating performance field cage test should be adjusted according to environmental conditions and fly activity (i.e. early start on hot days) and test duration should cover most of the known sexual activity period for the species under the local conditions. In the absence of such information, the test should cover most of the day. Ideally, tests should be run “blind”; i.e., technicians running the test should not be told which colour of marking corresponds to wild or sterile. In addition colors should be alternated randomly among replicates. 154

Data gathering

Collection of mating couples After release of females, observers should screen the tree for mating couples. This can be done continuously or a census of mating couples can be done under a certain frequency. Census frequency should be at least every 10-15 minutes, or even shorter for species that have shorter mating durations. Capture mating couples individually in the vials, taking care to get only one pair (and only 2 flies) per vial. Look at the pair carefully in order to confirm that copulation is taking place; males sometimes mount the female without real genital contact. If flies are not marked with paint but with fluorescent dye, keep them well identified (number each vial in accordance to the record is followed in the form) for a later recognition under the UV lamp or the fluorescent microscope.

Location of couples Write time the position on the tree (height, substrate, quadrant, and any information considered relevant) or any other place on the cage from where the couple is collected. Record all pertinent data, including time and type of male and female for each vial on the form. Label the vial accordingly, indicating the number assigned to the field cage in which the mating took place (if more than one cage is run on a given day), day of test and the number of the mating pair (couples should be numbered in the order they are collected in a given cage, starting from 1, for the first mating pair). This is extremely critical and attention should be paid in order not to mix the mating pairs.

Mating duration Write time when pairs were captured on the form. Once the couple is in the vial, care must be taken to ensure that the vials are kept in the shade and otherwise handled to minimize thermal or other stresses to the flies. Observe mating couples in vials on a regular basis (every 5 min or less) and note the time when uncoupling occurred.

Continue the test until the natural period of peak mating for that species under local conditions is well over. Once the test is over, with the aid of an aspirator remove the flies that did not mate from the cage. It is advisable to wash the tree to help removing any chemical signal left by the flies if the cage is to be used on the following or consecutive days.

In addition to the standard procedure described in the previous paragraphs, it is recommended to collect the following information:

Male calling time To determine the extent and timing of the males’ participation in pre-mating behaviours, a census may be taken at regular intervals (e.g., every 10-15 min or half hour). During the census, the number, location, and colour of mark are recorded for each male that is “calling”, or releasing pheromone within the cage or involved in courtship with female. Calling males are typically (but not always) on the underside of leaves. In medfly and in some Anastrepha species, they can be identified by the presence of what appears to be a drop of liquid on the tip of the abdomen (in reality, a sac is extended from the anus) and males also may inflate pouches on the pleura. Calling males may intermittently vibrate or “fan” their wings while standing in place; during fanning, anal sac is partially retracted and held under 155 the abdomen, and thus is more difficult to observe. These data can also be used to check diel periodicity of sexual behaviour among sterile males in comparison with wild males.

Incidence of remating This requires continuation of the test for a second or even more days. It is recommended then that this part is held under laboratory conditions to avoid dependency on weather conditions. Here again, care should be taken when collecting mating pairs during the first day to ensure that they are not disturbed to the point where they uncouple prematurely. Given that female behaviour is the core of this part, it is recommended that females are held singly in vials and two virgin males are offered every certain established period until females remate or the observation period ends. Remating rate and refractory period are the variables to be analyzed. The test should be run at leat twice the time of the mean refractory period (if known) and virgin males of the same population of the females should be offered at regular intervales (i.e. every day, every two days or three times a week). If possible, an ovisposition substrate can be provided to the females and the number of eggs laid recorded to be used as a covariable in the analysis. If female receptivity is very sensitive to fly density, then it is recomended to run the test under relaxed conditions in order to allow the females reject the males. For meaningful data, 25 females of each category should be analyzed.

Interpretation

Measures of sexual activity: For meaningful data, the basic mating performance field cage test (one-day observation) should be replicated ca. 6 - 10 times. Test replicates with more than ca. 25% of wild flies on the cage screen during calling periods may reflect inadequate environmental conditions (such as inadequate light, lack of water, leaves, etc.) and should therefore be repeated. In general it is suggested that fly activities occurring away from the host tree should not be included in the data analysis. This statement, however, should be taken with caution in compatibility tests given that the occurrence of leks and matings in the screen of the cage may also reflect some kind of spatial (i.e. ecological) isolation. For this reason it is recommended that any information should be recorded during the test and discarded if considered properly at the moment of data analysis.

Proportion of mating (PM) . The proportion of flies mating measures the suitability of the flies and the environment for mating and is defined as

PM = No. of pairs collected / No. of females released

Mean percentage of mating provides a useful indication of mating propensity. With C. capitata , mating propensity is considered adequate when 50% of flies from all combinations of strain and sex participate in mating. In practice, flies from some wild strains (especially the females) are more reluctant than sterile females to mate in field cages. At any rate, data from a cage should be discarded if less than 20% of both males and females from any strain participate in mating.

Indices of strain sexual compatibility: Several indices have been developed to quantify the sexual compatibility between strains/populations. The indices should be computed separately for each cage. Analysis of test results should involve the use of all of the main indices available (RII, ISI, FRPI, MRPI, see 156 bellow for a detailed description). It is advisable to include χ 2 Goodness of Fit analysis to assess statistically significant departure from random mating. .

Relative Isolation Index (RII) . The RII is a measure of mating compatibility between two strains. AA× BB RII = AB× BA Values of 1 indicates random mating between strains which is desirable in terms of SIT control; values greater than one indicate positive assortative mating, i.e., males from population A tends to mate with females from population A and vice versa (see graph below ). The RII has some advantages over other indices of compatibility being the most relevant to this type of tests the fact that it is not affected by the overall level of participation of the different types of flies, but only by whom they chose to mate.

SS× WW RII = SW× WS RII

0 +1 +10 +100 + Negative Random Positive assortative mating assortative mating (Sexual mating compatibility) (Sexual isolation) Graphic representation of the Relative Isolation Index (RII). The value shown represents the mean value obtained when comparing wild and sterile Ceratitis capitata flies in field cages.

However, RII also has some disadvantages. First, it is undefined if one of the two heterotypic crosses is zero. Second, when the number of matings in any one category is small, adding or subtracting a single mating in that category will cause a large change in the value of the index. Third, it can be difficult to normalize if the data are to be analysed statistically. Values of RII larger than 1 indicate that there is some difference in mating behaviour (in a broad sense) between the two populations, and that one or both strains are tending to mate assortatively (i.e., like with like). Values of RII that trigger corrective action will probably be found to vary from species to species, and wild flies from some areas seem to consistently produce higher RII ’s than flies from other areas.

Isolation Index (ISI). The ISI is a measure of mating compatibility.

(AA+ BB) − (AB + BA) ISI = AA+ BB + AB + BA

Its values range from -1 (complete negative assortative mating; i.e., all matings are with members of the opposite strain) through 0 (random mating) to +1 (complete positive assortative mating; total mating isolation of the two strains) (see graph below ). The main advantage of this index is that, by ranging from –1 to +1, it is easier to assess the deviation 157 from the expected value of 0 than it is with index ranging from 0 to infinity. Compared to the RII , ISI is not as sensitive to a change in a single mating and can always be defined, whatever are the values of the 4 types of mating. In general, values of ISI consistently larger than 0.5 suggest that some positive assortative mating took place (which should be explained by analysing the values of MRPI and FRPI ).

SS( + WW − SW() + )WS ISI = SS +WW + SW +WS ISI

-1 -0.75 -0.5 -0.25 0 0.25 0.5 0.75 +1 Negative Random mating Positive assortative (Sexual compatibility) assortative mating mating (Sexual isolation)

()()SS+ WS − SW + WW ()()SS+ SW − WS + WW FRPI = MRPI = SS+ SW + WS + WW SS+ SW + WS + WW

FRPI

MRPI

-1 -0.75 -0.5 -0.25 0 0.25 0.5 0.75 +1 All matings Equal mating All matings by wild propensity by sterile individuals individuals

Graphic representation of the Isolation Index (ISI) and of the Male and Female Relative Performance indices (MRPI and FRPI). These indices should only be considered together for a better understanding. The value shown was obtained when comparing wild and sterile Anastrepha ludens flies in field cages (after Hernandez et al. 2003).

Male Relative Performance Index (MRPI). The MRPI is a relative measure of mating propensity of males of one population versus males from the other.

(AA+ AB) − (BA+ BB) MRPI = AA+ AB + BA+ BB

A value of 1 indicates all matings in the cage were done by sterile males, and -1 indicates all mating was done by wild males. Zero indicates that wild and sterile males participated equally in mating. This index must be used in addition to the mean percentages of different types of flies participating in mating, and complements the ISI and FRPI (below).

158

Female Relative Performance Index (FRPI) . The FRPI is the counter part of the MRPI and serves as a measure of mating propensity for female flies.

(AA+ BA) − (AB + BB) FRPI = AA+ AB + BA+ BB

The joint analysis of ISI , MRPI and FRPI , should provide a complete and reliable picture of the sexual compatibility between strains and, should a deviation from the expected standard be encountered, the reasons why it occurred.

Mating time: The time at which matings take place is species and/or strain specific, as such in this type of test it is important to evaluate of this trait is affected or not by the origin of the other population present.

Copulation duration: Length of time spent in copula can be an indication of laboratory adaptation of a strain and can be related to transfer of sperm and accessory gland fluid to the female fly or the occurrence of some kind of guard posture. Duration of copulation can be compared among crosses by means of ANOVA or with non parametric statistics. In C. capitata , it has been noted that an exceptionally long duration of mating (>3 hours) very often resulted in no sperm being transferred to the female. However, short duration of mating for sterile males, relative to those of males mating with the same type of female, may be indicative of some kind of isolation, and may correlate with other related aspects, such as those on the incidence of remating.

Male calling time: Calling, or releasing pheromone to attract female flies, is an early and critical step in a male’s effort to secure a mate. The incidence of calling is a component of mating propensity, and a low incidence of calling could be indicative of low fly quality or vitality. In addition, location of calling males is relevant in this type of tests given that they may reflect the occurrence or not of mixed leks.

Female acceptance: Estimating the rate at which females accept males from their own origin or from the other requires more detailed observational studies, quantifying the number of female visits to the different types of calling and courting males, and recording rejection or acceptance of males by females. However, these data is of outmost importance in the case of incipient isolation between two origins.

Incidence of remating: Remating in wild tephritid females is more common in nature than previously believed. This behaviour can be interpreted as the outcome from what the female received during the first mating and Postcopulatory decision affected by her reproductive potential. Failings in the first election may be reverted in a second mating and in the case of compatibility studies it may reflect a rejection of the first male.

Modification to the mating performance field cage test Other variations of the mating performance field cage test may be conducted if deemed necessary. Examples of possible alternatives include: different ratios; use of various potted 159 host plants; use of higher male:female ratio by releasing females slowly over time (as would be expected in natural leks in the field); replacement of individuals as the couples are formed; etc. In the specific case of this cryptic species complexes CRP it is desirable to agree to which etent changes are appropriate or may impede comparisons between investigations. Use of tests with alternate designs may be particularly valuable in diagnosing causes of less-than- desirable levels of sexual compatibility but it is recommended to perform first the agreed protocol.

Literature

Aluja, M., I. Jacome, A. Birke, N. Lozada and G. Quintero. 1993. Basic patterns of behavior in wild Anastrepha striata (Diptera: Tephritidae) flies under field cage conditions. Ann. Entomol. Soc. Am. 86(6): 776-793. Baker, P.S. and A.S.T. Chan. 1991. Identification of tephritid fruit fly dispersal. Guidelines for a sterile release programme. J. Appl. Entomol. 112: 410-421. Baker, P.S., A.S.T. Chan and M.A. Jimeno-Zavala. 1986. Dispersal and orientation of sterile Ceratitis capitata and Anastrepha ludens (Tephritidae) in Chiapas. J. Appl. Ecol. 23: 27-38. Bateman, A. J. 1949. Analysis of data on sexual isolation. Evolution. 3: 174-177. Bloem, K., S. Bloem, D. Chambers and E. Muñiz. 1993. Field evaluation of quality: release-recapture of sterile medflies of different sizes. 295-296 In Aluja, M., and P. Liedo [eds.] Fruit flies: biology and management. Springer-Verlag, New York, NY, USA. Boller, E. F., U. Remund, B. I. Katsoyannos and W. Berchtold. 1977. Quality control in European cherry fruit fly: evaluation of mating activity in laboratory and field cage tests. Z. Angew. Ent. 83: 183-201. Briceño, R. D. and W. G. Eberhard. 1998. Medfly courtship duration: a sexually selected reaction norm changed by crowding. Ethology Ecology & Evolution. 10: 369-382. Briceño, R. D., W. G. Eberhard, J. C. Vilardi, P. Liedo and T. E. Shelly. 2002. Variation in the intermittent buzzing songs of male medflies (Diptera: Tephritidae) associated with geography, mass-rearing, and courtship success. Fla. Entomol. 85(1): 32-40. Briceño, R. D., D. Ramos and W. G. Eberhard. 1996. Courtship behavior of male medflies Ceratitis capitata (Diptera: Tephritidae) in captivity. Fla. Entomol. 79(2): 130-143. Burk, T. and J.C. Webb. 1983. Effect of male size on calling propensity, song parameters, and mating success in Caribbean fruit flies, Anastrepha suspensa (Loew) (Diptera: Tephritidae). Ann. Entomol. Soc. Am. 76: 678 Calcagno, G. E., F. Manso and J. C. Vilardi. 2002. Comparison of mating performance of medfly (Diptera, Tephritidae) genetic sexing and wild type strains: field cage and video recording experiments. Fla. Entomol. 85(1): 41-50. Calkins, C. O. and J. C. Webb. 1983. A cage and support framework for behavioral tests of fruit flies in the field. Fla. Entomol. 66(4): 512-514. Cayol, J. P., P. De Coronado and M. Taher. 2002. Sexual compatibility in medfly (Diptera, Tephritidae) from different origins. Fla. Entomol. 85(1): 51-57. 160

Cayol, J. P. 2000. Changes in sexual behavior and in some life history traits of Tephritid species caused by mass-rearing processes. 843-860. In Aluja, M. and A. Norrbom [eds.], Fruit Flies (Tephritidae): Phylogeny and Evolution of Behavior. CRC Press. Boca Raton, Florida. Cayol, J. P., J. Vilardi, E. Rial and M. T. Vera. 1999. New indices and method to measure the sexual compatibility and mating performance of medfly (Diptera, Tephritidae) laboratory reared strains under field cage conditions. J. Econ. Entomol. 92(1): 140-145. Chambers, D. L., C. O. Calkins, E. F. Boller, Y. Itô and R. T. Cunningham. 1983. Measuring, monitoring and improving the quality of mass-reared Mediterranean fruit flies, Ceratitis capitata (Wied). 2. Field tests for confirming and extending laboratory results. J. Appl. Ent. 95: 285-303. Dunham, M. 1994. An easily-constructed temporary cage for studying animals in the field. Fla. Entomol. 77(4): 505-508. Fried, M. 1971. Determination of sterile insect competitiveness. J. Econ. Entomol. 64: 869- 872. Hendrichs, J. 1986. Sexual selection in wild and sterile Caribbean fruit flies, Anastrepha suspensa . M.Sc.Thesis. Univ. of Florida, Gainesville, Fla. 263 pp. Hendrichs, J., V. Wornoayporn, B. I. Katsoyannos and K. Gaggl. 1993. First field assessment of the dispersal and survival of mass reared sterile Mediterranean fruit fly males of an embryonal temperature sensitive genetic sexing strain. 453-462. In IAEA [ed.], Management of insect pests: nuclear and related molecular and genetic techniques. IAEA. Vienna, Austria. Hernandez, E., D. Orozco, M. Hernandez, J. S. Meza, S. Flores and J. Dominguez. 2003. Quality assurance of mass produced and released fruit flies: third progress report. RC 10839/R2. IAEA. Vienna, Austria. Hibino, Y. and O. Iwahashi. 1989. Mating receptivity of wild type females for wild type males and mass-reared males in the Melon fly, Dacus cucurbitae Coquillett (Diptera: Tephritidae). Appl. Ent. Zool. 24(1): 152-154. Hibino, Y. and O. Iwahashi. 1991. Appearance of wild females unreceptive to sterilized males on Okinawa Island in the eradication program of the Melon fly, Dacus cucurbitae Coquillett (Diptera: Tephritidae). Appl. Ent. Zool. 26(2): 265-270. Jackson, C. G. and J. P. Long. 1997. Mating behavior of Bactrocera latifrons (Diptera: Tephritidae) in field cages. Ann. Entomol. Soc. Am. 90(6): 856-860. Jang, E. B., D. O. McInnis, D. R. Lance and L. A. Carvalho. 1998. Mating-induced changes in olfactory-mediated behavior of laboratory-reared normal, sterile, and wild female Mediterranean fruit flies (Diptera: Tephritidae) mated to conspecific males. Ann. Entomol. Soc. Am. 91(1): 139-144. Katsoyannos, B.I., N.T. Papadopoulos, J. Hendrichs and V. Wornoayporn. 1989. Comparative response to citrus foliage and citrus fruit odour by wild and mass-reared sterile Mediterranean fruit fly males of a genetic sexing strain. J. Appl. Ent. 123: 139- 143. Koyama, J. H. Nakamori and H. Kuba. 1986. Mating behavior of wild and mass-reared strains of the melon fly, Dacus cucurbitae Coquillett (Diptera: Tephritidae), in a field cage. Appl. Ent. Zool. 21(2): 203-209. 161

Kuba, H. and J. Koyama. 1985. Mating behavior of wild melon flies, Dacus cucurbitae Coquillett (Diptera: Tephritidae) in a field cage: courtship behavior. Appl. Ent. Zool. 20(4): 365-372. Lance, D. R., D. O. McInnis, P. Rendon and C. G. Jackson. 2000. Courtship among sterile and wild Ceratitis capitata (Diptera: Tephritidae) in field cages in Hawaii and Guatemala. Ann. Entomol. Soc. Am. 93(5): 1179-1185. Liedo, P., G. Ibarra, A. B. Davila, M. I. Barrios, M. L. Sosa and G. Perez-Lachaud. 1996. Mating competitiveness of three mass-reared strains of the Mediterranean fruit fly (Diptera: Tephritidae). In IAEA [ed.], Second RCM on “Medfly mating behaviour under field cage conditions”, Tapachula, Mexico, 19-23 February 1996. IAEA. Vienna, Austria. Liedo, P., E. De Leon, M. I. Barrios, J. F. Valle-Mora and G. Ibarra. 2002. Effect of age on the mating propensity of the Mediterranean fruit fly (Diptera: Tephritidae). Fla. Entomol. 85(1): 94-101. Lux, S. A., F. N. Munyiri, J. C. Vilardi, P. Liedo, A. Economopoulos, O. Hasson, S. Quilici, K. Gaggl, J. P. Cayol and P. Rendon. 2002. Consistency in courtship pattern among populations of medfly (Diptera: Tephritidae): comparisons among wild strains and strains mass reared for SIT operations. Fla. Entomol. 85(1): 113-125. McInnis, D. O., D. R. Lance and C. G. Jackson. 1996. Behavioral resistance to the Sterile Insect Technique by Mediterranean fruit fly (Diptera: Tephritidae) in Hawaii. Ann. Entomol. Soc. Am. 89(5): 739-744. McInnis, D. O., P. Rendon, E. B. Jang, A. Van Sauers-Muller, R. L. Sugayama and A. Malavasi. 1999. Interspecific mating of introduced, sterile Bactrocera dorsalis with wild B. carambolae (Diptera: Tephritidae) in Suriname: a case for cross-species sterile insect technique. Ann. Entomol. Soc. Am. 758-765. McInnis, D. O., P. Rendon and J. Komatsu. 2002. Mating and remating of medflies (Diptera: Tephritidae) in Guatemala: individual fly marking in field cages. Fla. Entomol. 85(1): 126-137. Orozco, D. and R.O. Lopez. 1993. Mating competitiveness of wild and laboratory mass- reared medflies: effect of male size. 185-188. In Aluja, M., and P. Liedo, [eds.], Fruit flies: biology and management. Springer-Verlag, New York, NY, USA. Plant, R.E. and R.T. Cunningham. 1991. Analyses of the dispersal of sterile Mediterranean fruit flies (Diptera: Tephritidae) released from a point source. Environ. Entomol. 20: 1493-1503. Prokopy, R. J. and J. Hendrichs. 1979. Mating behavior of Ceratitis capitata on a field- caged host tree. Ann. Entomol. Soc. Am. 72: 642-648. Rendon, P., D. O. McInnis, D. R. Lance and J. Stewart. 2000. Comparison of medfly male-only and bisexual releases in large scale field trials. 517-525. In Tan, K. H. [ed.], Area-wide management of fruit flies and other major insect pests. Penerbit Universiti Sains Malaysia. Penang, Malaysia. Robinson, A. S., U. Cirio, G. H. S. Hooper and M. Capparella. 1986. Field cage studies with a genetic sexing strain in the Mediterranean fruit fly, Ceratitis capitata . Entomol. Exp. Appl. 41: 231-235. Shelly, T. E., S. S. Kennelly and D. O. McInnis. 2002. Effect of adult diet on signaling activity, mate attraction, and mating success in male Mediterranean fruit flies (Diptera: Tephritidae). Fla. Entomol. 85(1): 150-155. 162

Sivinski, J. M., C. O. Calkins and R. Baranowski. 1994. A container for eclosion and holding adult insects prior to release. Fla. Entomol. 77(4): 513-515. Stalker, H. D. 1942. Sexual isolation studies in the species complex Drosophila virilis . Genetics. 27: 238-267. Vera, M. T., R. J. Wood, J. L. Cladera and A. S. Gilburn. 2002. Factors affecting female remating frequency in the Mediterranean fruit fly (Diptera: Tephritidae). Fla. Entomol. 85(1): 156-164. Wong, T. T. Y., J. I. Nishimoto and H. Melvin Couey. 1983. Mediterranean fruit fly (Diptera: Tephritidae): further studies on selective mating response of wild and of unirradiated and irradiated, laboratory-reared flies in field cages. Ann. Entomol. Soc. Am. 76(1): 51-55. Zapien, G. I., J. Hendrichs, P. Liedo and A. Cisneros. 1983. Comparative mating behaviour of wild and mass-reared sterile medfly Ceratitis capitata (Wied.) on a field cage host tree - II. Female mate choice. 397-409. In Cavalloro, R. [ed.], Fruit flies of economic importance. A.A. Balkema. Rotterdam, the Netherlands.

M. Teresa Vera / July, 2010 for IAEA CRP

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Appendix 7: Male Sex Pheromonal Components in Bactrocera Species – Extraction of Rectal (Pheromonal) Gland for Chemical Analyses

Jacentkovski in 1932 first demonstrated the existence of insect sex pheromone by using traps containing virgin gypsy moth females, which attracted and trapped many males, placed in gypsy moth infested woods. Only till 1959, Butenandt and coworkers after extracting from about half a million female silkworms, Bombyx mori , reported the first sex pheromone 'bombykol' was identified using traditional chemistry. We have come a long way since then, with the help of highly sensitive instrumentation and recording techniques, such as the gas chromatography-mass spectrophotometry ( GC-MS) coupled to an electro-anntenograph (EAG), a scientist can now extract and identify an insect's sex pheromone from a few individuals. In the case of fruit flies, particularly those from the genus Bactrocera, ( unlike the lepidopterans) males produce sex pheromone at sexual maturity. For many species, males make use of external sources of chemical (attractant) to produce sex pheromonal component(s).Therefore, a single male may be sufficient to confirm identity of the sex pheromonal component(s) after feeding on the specific attractant, provided proper procedure is followed.

All Bactrocera species may be categorized into three groups based on their non-response (28 species confirmed and 258 species listed under “lures unknown”) or response to two very potent attractants – cue-lure (195 species) and methyl eugenol (ME) (ca 84 species) (IAEA 2000, 2005). Approximately a dozen of the ME responsive/sensitive species of Bactrocera including several putative sibling species of the Bactrocera dorsalis complex, such as B. carambolae Drew & Hancock , B. dorsalis (Hendel), B. invadens Drew, Tsuruta & White, B. occipitalis (Bezzi), B. papayae Drew & Hancock , and B. philippinensis Drew & Hancock form a group of serious polyphagous pests that currently cause high economic losses in the production of fruits. They are also very important quarantine pests that may interfere or disrupt international free and fair trade of fresh fruits and vegetables between exporting and importing countries globally.

Credit must be given to Howlett who first demonstrated that male tephritid flies (Dacus spp.) were attracted to citronella oil in in 1912. He subsequently showed that the flies were actually attracted to ME in 1915. Since then, there were much speculations as to the actual role of ME in tephritid flies.

Males of B. dorsalis and B. papayae are strongly attracted to and compulsively feed on the ME which acts as a sex pheromone precursor that is converted to two major components - E- coniferyl alcohol and 2-allyl-4,5-dimethoxyphenol (Nishida et al. 1988; Tan and Nishida 1996, 1998). Nonetheless, the ME can also act as a booster component to complement endogenously produced sex pheromone as in B. carambolae in which it is converted only to E-coniferyl alcohol (Tan and Nishida 1996; Wee and Tan 2007). Table 1 shows the male sex pheromone components of several Bactrocera species after feeding on an attractant ME or raspberry ketone in the case of the melon fly B. cucurbitae. In the male B. dorsalis/papayae , the consumed ME is biotransformed in the crop and its metabolites are then transported via the haemolymph to the rectal gland (Hee and Tan 2006), where they are sequestered via rectal papillae and temporarily stored before being released as sex pheromone (Fig. 1) (Khoo and Tan 2005).

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Native males seek and ingest ME from natural sources (Tan 2009; Tan et al. 2002, 2006). There are ca 350 plant species belonging to 61 families that possess ME, in varying quantities from trace amounts to >90% of essential oils, as a constituent component and/or release as a component of floral fragrance (Tan, compiled list unpublished). Consumption of the ME has been shown to improve significantly male mating performance and competitiveness of B. dorsalis (Shelly and Dewire 1994, 2000; Tan and Nishida 1996), B. papayae (Tan and Nishida 1996, 1998) B. carambolae (Wee et al. 2007), B. correcta (Orankanok et al. 2009) and B. zonata (Quilici et al. 2004; Sookar et al. 2009).

Table 1. Sex pheromone after feeding on methyl eugenol or raspberry ketone

B. carambolae - E-coniferyl alcohol (CF) (endogenously produced - 6-oxo-1-nonanol + N- 3 methylbutyl acetamide + 1,6-nonanediol + ethyl benzoate)

B. dorsalis - 2-allyl-4,5-dimethoxyphenol (DMP) + CF + 3,4-dimethoxycinnamyl alcohol (DCA)

B. papayae - DMP + CF + DCA

B. umbrosa - DMP + DCA + 3,4-dimethoxy-hydroxyallyl-benzene

B. cucurbitae - Raspberry ketone (endogenously produced - 1,3-nonanediol + methyl, ethyl & propyl 4-hydroxybenzoate + N-3-methylbutyl methoxyacetamide)

Figure 1. Male rectal gland of B. dorsalis showing the presence of auto-fluorescent compounds during sequestration of sex pheromonal chemicals from the haemolymph at 10 to120 minutes after feeding on methyl eugenol. (Adapted from Khoo and Tan (2005). White arrows – the chemicals detected within 1 hour.

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10 min 40 min

20 min 2 hours

Figure 2. Male rectal glands extracted from sexually mature males (15 days old) – deprived of ME (left) and fed with ME ( right - one day post-feeding)

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With highly sensitive instrumentation such as the GC-MS that uses fine capillary column to analyze sex pheromonal components, it is very important that samples injected into the column must not contain contaminants (especially non-volatile compounds and macromolecules ) that will clog up the column. It is also important to note that sex pheromonal compounds are usually volatile and exist in microgram or submicrogram levels. Therefore, it is pertinent to follow proper rectal gland extraction procedure, before preserving it for future chemical analyses.

1. Solvent for extraction preferably pure ethanol – redistilled (N.B. This solvent is very hygroscopic - so do not expose to atmospheric air for long periods, especially in the humid tropics). If this solvent is not available, then, absolute alcohol (ethanol) is suitable (N.B. Please provide a 1 ml sample for solvent check for possible impurities). 2. Prepare glass vials (1 or 2 ml with teflon lined screw-caps) for preservation of gland – fill each vial with 250 microlitre (0.25 ml) of the solvent and tighten the screw cap until use. (N.B. One vial for preservation of one rectal gland). 3. Fine pointed forceps – Firstly, wash and clean with a detergent; and secondly, rinse the tips with a few drops of the solvent just before use. 4. Immobilize a male fly, preferably without the use of carbon dioxide, by transferring the fly into a small clean plastic bag or cleaned container, making sure that the fly cannot escape, and then place it in either a refrigerator for 20-30 minutes at < 15 o C or a freezer < - 5o C for 1-2 minutes. 5. Rectal gland removal - Place the immobilized fly ventral side up on the stage of a dissecting microscope (Fig. 3). One may hold the fly in place either with a pair of forceps or with one's fore finger and thumb. Then, with a pair of cleaned fine pointed forceps grip firmly the base of the aedaegus (Fig. 3A), and simultaneously pull it gently away from the fly's abdomen until the rectal gland is completely exposed (Fig. 3B & C) – due care should be taken, by avoiding sudden jerks, to ensure that the rectal sac does not burst especially when it is bloated. Then cut the hind gut at approximately 1 mm from the rectal gland (Fig 3 D). The freshly removed gland (Fig. 4) on the forceps tip is immediately introduced into a vial containing pure alcohol. After that, the teflon lined screw-cap is replaced and tightened (finger tight) to the vial. To avoid possible leakage during transportation (especially drastic climatic changes), the narrow gap between rim circumference of the screw-cap and the glass vial is tightly sealed by simultaneously stretching and wrapping a parafilm strip (ca 1.5 cm x 5 cm) several times around it . After labeling the vial, store the extracted rectal gland in a refrigerator until dispatch for chemical analyses. 6. For comparison of sexually mature flies fed with and deprived of of an attractant, e.g. methyl eugenol (ME) – item 5 needs to be repeated for rectal gland removal from the ME-fed and the ME-deprived (as controls) flies.

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Figure 3. Removal of rectal (pheromone) gland under magnification. Steps A-D.

A B

C D

Figure 4. Freshly removed male rectal gland and associated aedeagal apparatus.

References

Butenandt, A., Beckmann, R., Stamm, D. and Hecker, E. 1959. Uber den Sexual-Lockstaff des Seidenspinners Bombyx mori . Reindarstellung und Konstitution. Z. Naturforsch. B, 14: 283-284. Hee A.K.H. and Tan K.H. 2006. Methyl eugenol-derived sex pheromonal components transport in the male fruit fly, Bactrocera dorsalis . Comp. Biochem. Physiol. Part C, 143: 422-428. Howlett F.M. 1912. VII. The effect of oil of Citronella on two species of Dacus . Tran Entomol Soc Lond 1912:412-418. Howlett F.M. 1915. Chemical reactions of fruit-flies. Bull Entomol Res 6:297-305. 168

IAEA 2000. Action Plan – Peach Fruit Fly, Bactrocera zonata (Saunders). Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, International Atomic Energy Agency, Vienna. 50 pp. IAEA 2003. Trapping guidelines for area-wide fruit fly programmes. Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, International Atomic Energy Agency, Vienna. 47 pp. Jacentkovski, J.A. 1932. Nonnen kontroll- und Vorbeugunsmethode nach Professor Forst.-Ing. Ant. Dyk. Anz. Schädlingskunde,14: 52 and 65. Khoo C.C.H. and Tan K.H. 2005. Rectal gland of Bactrocera papayae : ultrastructure, anatomy and sequestration of auto fluorescent compounds upon methyl eugenol consumption by the male fly. Micros Res Tech 67:219-226. Nishida R., Tan K.H., Serit M., Lajis N.H., Sukari A.M., Takahashi S., Fukami H. 1988. Accumulation of phenylpropanoids in the rectal glands of males of the Oriental fruit fly, Dacus dorsalis . Experientia 44:534-536. Orankanok W., Chinvinijkul S., Sawatwangkhoungm A., Pinkaew S., and Orankanok S. 2009. Application of chemical supplements to enhance Bactrocera dorsalis and B. correcta sterile males performance in Thailand. Fourth FAO/IAEA Research Co-ordination meetings on “Improving Sterile Male Performance in Fruit Fly SIT Programmes” 21-25 September 2009, Péreybère, Mauritius. Quilici S., Duyck P.F., and Franck A. 2004. Preliminary experiments on the influence of exposure to methyleugenol on mating success of males in the peach fruit fly, Bactrocera zonata . In: 1st RCM on improving sterile male performance in fruit fly SIT, Antigua, Guatemala, 25 to 29 October 2004. Shelly T.E. and Dewire A-L.M. 1994. Chemically mediated mating success in male Oriental fruit flies (Diptera: Tephritidae). Ann Entomol Soc Am 87:375-382. Shelly T.E. and Dewire A-L.M. 2000. Flower-feeding affects mating performance in male Oriental fruit flies (Diptera: Tephritidae). Ecol Entomol 25:109-114. Sookar P., Alleck M., Ahseek N., Khayrattee F.B., and Permalloo S. 2009. Improving male reproductive performance of Bactrocera zonata and Bactrocera cucurbitae . Fourth FAO/IAEA Research Co-ordination meetings on “Improving Sterile Male Performance in Fruit Fly SIT Programmes” 21-25 September 2009, Péreybère, Mauritius. Tan K.H. 2009. Fruit fly pests as pollinators of wild orchids. Orchid Digest 73:180-187. Tan K.H. and Nishida R. 1996. Sex pheromone and mating competition after methyl eugenol consumption in the Bactrocera dorsalis complex. In: McPheron BA, Steck GJ (eds) Fruit fly Pests – A world assessment of their biology and management. St. Lucid Press, Florida, pp 147-153. Tan K.H. and Nishida R. 1998. Ecological significance of male attractant in the defence and mating strategies of the fruit fly, Bactrocera papayae . Entomol Exp Appl 89:155-158. Tan KH, Nishida R, and Toong YC. 2002. Floral synomone of a wild orchid, Bulbophyllum cheiri , lures Bactrocera fruit flies for pollination. J Chem Ecol 28:1161-1172. Tan K.H. Tan L.T., and Nishida R. 2006. Floral phenylpropanoid cocktail and architecture of Bulbophyllum vinaceum orchid in attracting fruit flies for pollination. J Chem Ecol 32:2429- 2441. Wee S.L. and Tan K.H. 2007. Temporal accumulation of phenylpropanoids in male fruit flies, Bactrocera dorsalis and B. carambolae (Diptera: Tephritidae) following methyl eugenol consumption. Chemoecology 17:81-85.

K.H. Tan/13 th July, 2010 for IAEA CRP

169

Appendix 8: List of References

Adsavakulchai A, Baimai V, Prachyabrued W, Grote PJ, Lertlum S. 1999. Morphometric study for identification of the Bactrocera dorsalis complex (Diptera: Tephritidae) using wing image analysis. Biotropica 13: 37–48

Aketarawong N, Bonizzoni M, Thanaphum S, Gomulski LM, Gasperi G, Malacrida AR, Gugliemino CR. 2007. Inferences on the population structure and colonization process of the invasive oriental fruit fly, Bactrocera dorsalis (Hendel). Molecular Ecology 16 : 3522-3532.

Allwood AJ, Leblanc L. 1996. Losses Caused by Fruit Flies (Diptera: Tephritidae) in Seven Pacific Island Countries. In: Management of Fruit Flies in the Pacific: A regional symposium, Nadi, Fiji 28–31 October 1996. Eds: A.J. Allwood and R.A.I. Drew, pp 208-211.

Aluja, M, Perez-Staples D, Macias Ordoñez R, Piñero J, McPheron B, Hernandez Ortiz V. 2003. Nonhost status of Citrus sinesis Cultivar Valencia and C. paradisi Cultivar Ruby Red to Mexican Anastrepha fraterculus (Diptera: Tephritidae). J. Econ. Entomol. 96 (6): 1693-1703.

Armstrong KF, Cameron CM, Frampton ER. 1997. Fruit fly (Diptera: Tephritidae) species identification: a rapid molecular diagnostic technique for quarantine application. Bulletin of Entomological Research 87 : 111-118.

Armstrong KF, Cameron CM . 2000. Species identification of Tephritids across a broa d taxonomic range using ribosomal DNA. In K.H. Tan (ed.) Area -wide control of fruit flies and other insect pests, pp 703 - 710.

Armstrong KF, Ball SL. 2005. DNA barcodes for biosecurity: invasive species identification. Philosophical Transactions of the Royal Society London 360 : 1462, 1813- 1823.

Baimai V, Phinchongsakuldit J, Tigvattananont S. 1999. Metaphase karyotypes of fruit flies of Thailand. IV. Evidence for six new species of the Bactrocera dorsalis complex. Cytologia 64 : 371–377.

Břízová R, Mendonça AL, Vanícková L, Alana L. Mendonça AL, Da Silva CE, Tomčala A, Jordão Paranhos B, Simões Dias V, Joachim-Bravo I, Hoskovec M, Kalinová B, Do Nascimento RR. 2013. Pherormone analyses of the Anastrepha fraterculus (Diptera: Tephritidae) cryptic species complex. Fla. Entomol. 96: 11-7-1115.

Baimai V, Sumrandee C, Tigvattananont S, Trinachartvanit W. 2000. Metaphase karyotypes of fruit flies of Thailand. V. Cytotaxonomy of ten additional new species of the Bactrocera dorsalis complex. Cytologia 65 : 409–417.

Baimai V, Trinachartvanit W, Tigvattananont S, Grote PJ, Poramarcom R, Kijchalao U. 1995. Metaphase karyotypes of fruit flies of Thailand. I. Five sibling species of the Bactrocera dorsalis complex. Genome 38 : 1015–1022. 170

Barnes BN. 2000. Fruit flies of economic importance in South Africa: an overview. In: Proceedings of the Indian Ocean Commission Regional Fruit Fly Symposium , 5–9 June 2000, Flic en Flac, Mauritius (eds Price NS, Seewooruthun I), pp. 101–107. Commission de l’ocèan Indien, Quatre Bornes, Mauritius.

Bezzi M. 1920. Notes on Ethiopian fruit-flies of the family Trypaneidae, other than Dacus . Bulletin of Entomological Research , 10 : 211–272.

Boykin, L. M., M. K. Schutze, M. N. Krosch, A. Chomic, T. A. Chapman, A. Englezou, K. F. Armstrong, A. R. Clarke, D. Hailstones, and S. L. Cameron. 2013. Multi-gene phylogenetic analysis of the south-east Asian pest members of the Bactrocera dorsalis species complex (Diptera: Tephritidae) does not support current taxonomy. Journal of Applied Entomology:DOI: 10.1111/jen.12047.

Cáceres, C., Segura, D. F., Vera, M. T., Wornoayporn, V., Cladera, J. L., Teal, P., Sapountzis, P., Bourtzis, K., Zacharopoulou, A. & Robinson, A. S. 2009. Incipient speciation revealed in Anastrepha fraterculus (Diptera; Tephritidae) by studies on mating compatibility, sex pheromones, hybridisation and cytology. Biological Journal of the Linnean Society 97 : 152–165.

Castañeda MR, Osorio A, Canal NA, Galeano PE. 2010. Species, distribution and hosts of the genus Anastrepha Schiner in the Department of Tolima, Colombia. Agronomía Colombiana 28 (2), 265-271.

Chua TH, Song BK, Chong YV. 2010. Development of Allele-Specific Single-Nucleotide Polymorphism-Based Polymerase Chain Reaction Markers in Cytochrome Oxidase I for the Differentiation of Bactrocera papayae and Bactrocera carambolae (Diptera: Tephritidae). Journal of Economic Entomology, 103(6):1994-1999.

Clarke AR, Armstrong KF, Carmichael AE, Milne JR, Raghu S, Roderick GK, Yeates DK. 2005. Invasive phytophagous pests arising through a recent tropical evolutionary radiation: the Bactrocera dorsalis complex of fruit flies. Annual Review of Entomology 50 : 293–319.

Delomen MLC, Mendioro MS, Diaz MGQ. 2013. Morphometric Analysis and DNA Barcoding of Fruit Flies Bactrocera occipitalis (Bezzi) and B. philippinensis Drew and Hancock (Diptera: Tephritidae) from Cavite and Davao del Norte. Philippine Journal of Science 142: 69-76.

De Meyer M. 2001a. Distribution patterns and host–plant relationships within the genus Ceratitis MacLeay (Diptera: Tephritidae) in Africa. Cimbebasia 17 : 219–228.

De Meyer M. 2001b. On the identity of the Natal fruit fly Ceratitis rosa Karsch (Diptera, Tephritidae). Bull. Inst. Roy. Sci. Nat. Belg. Entomol . 71 : 55–62.

Dhillon MK, Singh R, Naresh JS, Sharma HC. 2005. The melon fruit fly, Bactrocera cucurbitae : A review of its biology and management. Journal of Insect Science 5: 40-56.

171

Drew RAI. 1989. The tropical fruit flies (Diptera: Tephritidae: Dacinae) of the Australasian and Oceanian Regions. Memoirs of the Queensland Museum 26 : 1-521.

Drew RAI, Hancock DL. 1994. The Bactrocera dorsalis complex of fruit flies (Diptera: Tephritidae: Dacinae) in Asia. Bulletin of Entomological Research Supplement Series , Supplement No. 2, 68 p.

Drew RAI, Tsuruta K, White IM. 2005. A new species of pest fruit fly (Diptera: Tephritidae: Dacinae) from Sri Lanka and Africa. African Entomology 13 : 149-154.

Drew, RAI, Raghu S, Halcoop P. 2008. Bridging the morphological and biological species concepts: studies on the Bactrocera dorsalis (Hendel) complex (Diptera: Tephritidae: Dacinae) in South-east Asia. Biological Journal of the Linnean Society 93 : 217–226.

Drew RAI, Romig MC. 2013. Tropical Fruit Flies (Tephritidae: Dacinae) of South-East Asia. CAB International, Croydon, UK.

Dutra VS, Ronchi-Telles B, Steck GJ, Silva GJ . 2011. Egg morphology of Anastrepha spp. (Diptera: Tephritidae) in the fraterculus group using scanning electron microscopy. Ann. Entomol. Soc. Am. 104 (1): 16-24.

Dutra VS, Ronchi-Teles B, Steck GJ, Silva GJ. 2012. Description of larvae of Anastrepha spp. (Diptera: Tephritidae) in the fraterculus group. Annals Entomological Society of America 105: 529-538.

EPPO/CABI. 1997. Quarantine Pests for Europe , 2nd edition (eds Smith IM, McNamara DG, Scott PR, Holderness M). CAB International, Wallingford, UK.

FAO/IAEA/USDA. 2003. Manual for Product Quality Control and Shipping Procedures for Sterile Mass-Reared Tephritid Fruit Flies, Version 5.0. International Atomic Energy Agency, Vienna, Austria. 85 pp.

Fletcher BS. 1989. Life history strategies of tephritid fruit flies. In: Fruit Flies: Their Biology, Natural Enemies and Control , Vol. 3B (eds Robinson AS, Hooper GH), pp. 195– 208. Elsevier, Amsterdam, Netherlands.

Goday C, Selivon D, Perondini ALP, Graciano PG, Ruiz MF. 2006. Cytological characterization of sex chromosomes and ribosomal DNA location in Anastrepha species (Diptera: Teprhitidae). Cytogenetic and Genome Research 114 : 70-76.

Gomes Silva J. 2000. Estudos moleculares, pp. 29-39. In A. Malavasi and R. A. Zucchi [eds.], Moscas-das-frutas de importáncia econômica no Brasil. Conhecimento básico e aplicado. Holos Editora, Riber, Brazil.

Han H-Y, McPheron BA. 1994. Phylogenetic study of selected tephritid flies (Insecta: Diptera: Tephritidae) using partial sequences of the nuclear 18S ribosomal DNA. Biochem. Syst. Ecol. 22 : 447–457.

172

Handler AM, Zimowska GJ, Armstrong KF. 2008. Highly similar piggy Bac elements in Bactrocera that share a common lineage with elements in noctuid moths. Insect Molecular Biology 17 (4): 387–393.

Han H-Y, McPheron BA. 1997. Molecular phylogenetic study of Tephritidae (Insecta: Diptera) using partial sequences of the mitochondrial 16S ribosomal DNA. Mol. Phylogenet. Evol. 7: 17–32.

Hardy, D. E. 1969. Taxonomy and distribution of the oriental fruit fly and related species (Tephritidae-Diptera). Proceedings of the Hawaiian Entomological Society 20:395-428.

Hasanuzzaman M. 1998. Genetic studies on isozyme variations by electrophoresis and melanization test in the melon fly Bactrocera cucurbitae (Coq.) (Diptera: Tephritidae), with reference to irradiation treatment. M. Sc. Thesis, Department of Zoology, Faculty of Biological Science, University of Dhaka, Bangladesh, pp. 1-83.

Hasanuzzaman M, Yesmin F, Shahjahan RM, Begum A. 2003. Effects of gamma radiation on melanization in the larvae of melon fly, Bactrocera cucurbitae (Coq.) (Diptera: Tephritidae). Dhaka Univ. J. Biol. Sci., 12 (2): 123- 129.

Hasanuzzaman M, Idris AB. 2011. Comparative study on esterase isozyme patterns in the adult fly of Bactrocera papayae and B. carambolae (Diptera: Tephritidae). Proceedings of the MAPPS (Malaysian Plant Protection Society) National Seminar, Palm Garden Hotel IOI Resort, Putrajaya, Malaysia, 08 Dec, 2011, pp. 39-41.

Hasanuzzaman M, Idris AB. 2011. Esterase Isozyme Patterns during Development in Bactrocera papayae Drew and Hancock. Abstract, International Symposium on Tropical and Subtropical Fruits, The Imperial Mae Ping Hotel, Chiang Mai, Thailand, 19-22 Dec, 2011, pp. 38.

Haymer DS, Tanaka T, Teramae C. 1994. DNA probes can be used to discriminate between tephritid species at all stages of the life cycle (Diptera: Tephritidae). J. Econ. Entomol. 87 : 741–46.

He M, Haymer DS. 1997. Polymorphic intron sequences detected within and between populations of the oriental fruit fly (Diptera:Tephritidae). Ann. Entomol. Soc. Am. 90 : 825– 831

Hee A, Tan KH . 1998. Attraction of female and male Bactrocera papayae to conspecific males fed with methyl eugenol and attraction of females to male sex pheromone components. Journal of Chemical Ecology : 24 (4): 753-764.

Hee AKW, Tan KH. 2004. Male sex pheromonal components derived from methyl eugenol in the haemolymph of fruit fly Bactrocera papayae . Journal of Chemical Ecology. 30 : 2127- 2138.

Hee AKH, Tan KH . 2005. Transportation of methyl eugenol-derived sex pheromonal components in male fruit fly, Bactrocera dorsalis. Bulletin of Entomological Research. 95: 615–620.

173

Hee AKH, Tan KH. 2006. Methyl eugenol-derived sex pheromonal components transport in the male fruit fly, Bactrocera dorsalis . Comparative Biochemistry and Physiology Part C 143 : 422-428.

Hernández VH, Gomez Anaya JA, Sanchez S, McPheron BA, Aluja M. 2004. Morphometric analysis of Mexican and South American populations of the Anastrepha fraterculus complex (Diptera: Tephritidae) and recognition of a distinct Mexican morphotype. Bulletin of Entomol Research 94: 487 -499.

Hernández-Ortiz V., Bartolucci AF, Morales-Valles P, Frias D, Selivon D. 2012. Cryptic Species of the Anastrepha fraterculus Complex (Diptera: Tephritidae): A Multivariate Approach for the Recognition of South American Morphotypes. Ann. Entomol. Soc. Am. 105 (2): 305-318;

Iwahashi O. 1999. Distinguishing between the two sympatric species Bactrocera carambolae and B. papayae (Diptera: Tephritidae) based on aedeagal length. Ann. Entomol. Soc. Am. 92 : 639–643.

Iwahashi O. 1999. Distinguishing between two sympatric species Bactrocera occipitalis and B. philippinensis (Diptera: Tephritidae), based on aedeagal length. Ann. Entomol. Soc. Am. 92 : 182–187.

Iwahashi O. 2000. Speciation of Bactrocera dorsalis complex based on aedeagus length. In: Tan KH, ed. 2000. Area-Wide Control of Fruit Flies and Other Insect Pests . Penang, Malaysia: CABI Publ. 780 pp., pp. 591–94.

Iwahashi O. 2001. Aedeagal length of the oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), and its sympatric species in Thailand and the evolution of a longer and shorter aedeagus in the parapatric species of B. dorsalis . Appl. Entomol. Zool. 36 : 289–97.

Iwaizumi R, Kaneda M, Iwahashi O. 1997. Correlation of length of terminalia of males and females among nine species of Bactrocera (Diptera, Tephritidae) and differences among sympatric species of B. dorsalis complex. Ann. Entomol. Soc. Am. 90 : 664–666.

Jacquard C, Virgilio M, David P, Quilici S, De Meyer M, Delatte H. 2013. Population structure of the melon fly, Bactrocera cucurbitae , in Reunion Island. Biological invasions DOI 10.1007/s10530-012-0324-8.

Khamis, F. M., D. K. Masiga, S. A. Mohamed, D. Salifu, M. d. Meyer, and S. Ekesi. 2012. Taxonomic identity of the Invasive fruit fly pest, Bactrocera invadens: Concordance in morphometry and DNA barcoding. PLoS ONE:7(9) e44862. doi:44810.41371/journal.pone.0044862.

Khoo CCH, Tan KH. 2000. Attraction of female Bactrocera papayae to sex pheromone components with two different release devices. Journal of Chemical Ecology 26 : 2487-2496.

Krosch, M. N., M. K. Schutze, K. F. Armstrong, Y. Boontop, L. M. Boykin, T. A. Chapman, A. Englezou, S. L. Cameron, and A. R. Clarke. 2013. Piecing together an integrative taxonomic puzzle: microsatellite, wing shape and aedeagus length analysis of 174

Bactrocera dorsalis s.l. (Diptera: Tephritidae) find no evidence of multiple lineages in a proposed contact zone along the Thai/Malay Peninsula. Systematic Entomology 38: 2-13.

Lopes GN, Arias OR, Consoli FL, Zucchi RA. 2013. The Identity of Specimens of the Anastrepha fraterculus Complex (Diptera, Tephritidae) with Atypical Aculeus Tip. Neotropical Entomology 42: DOI 10.1007/s13744-013-0162-0.

Leblanc L, Hossain A, Khan SA, San Jose M, Rubinoff D. 2013. A Preliminary Survey of the Fruit Flies (Diptera: Tephritidae: Dacinae) of Bangladesh. Proceedings of the Hawaiian Entomological Society 45:51–58.

Leopold, RA. 2005. Colony maintenance and mass-rearing: using cold storage technology for extending the shelf-life of insects, pp. 100–102. In Book of Extended Synopses. FAO/IAEA International Conference on Area-Wide Control of Insect Pests: Integrating the Sterile Insect and Related Nuclear and Other Techniques, 9–13 May 2005, Vienna, Austria. IAEA-CN-131/49. IAEA, Vienna, Austria.

Li Y, Wu Y, Chen H, Wu J, Li Z. 2012. Population structure and colonization of Bactrocera dorsalis (Diptera: Tephritidae) in China, inferred from mtDNA COI Sequences. J. Appl. Entomol. 136: 241–251.

Ludeña B, Bayas R, Pintaud JC. 2011. Phylogenetic relationships of Andean-Ecuadorian populations of Anastrepha fraterculus (Wiedemann 1830) (Diptera: Tephritidae) inferred from COI and COII gene sequences. Ann. Soc. Entomol. Fr. 46 (3–4) : 344-350.

Mahmood K. 1999. Verification of specific status of pest species allied to Bactrocera dorsalis (Hendel) (Diptera: Tephritidae) using allozyme electrophoresis. Pakistan J. Zool. 31 : 159–165.

Malacrida AR, Guglielmino CR, D’Adamo P, Torti C, Marinoni F, Gasperi G. 1996. Allozyme divergence and phylogenetic relationships among species of tephritid flies. Heredity 76 : 592–602.

Malavasi, A, Morgante JS. 1982. Genetic variation in natural populations of Anastrepha (Diptera: Tephritidae). Rev. Bras. Genet. 5: 263-278.

McPheron BA, Han H-Y, Silva JG, Norrbom AL. 1999. Phylogeny of the genera Anastrepha and Toxotrypana (Trypetinae: Toxotrypanini) based upon 16S rRNA mitochondrial DNA sequences, pp. 343-361. In M. Aluja and A.L. Norrbom (eds.) Fruit flies (Tephritidae): phylogeny and evolution of behavior. Boca Raton, Florida, CRC Press, USA.

Morgante JS, Malavasi A, Bush GL. 1980. Biochemical systematics and evolutionary relationships of neotropical Anastrepha. Ann. Entomol. Soc. Am. 73 : 622-630.

Muraji M, Nakahara, S. 2002. Discrimination among pest species of Bactrocera (Diptera: Tephritidae) based on PCR-RFLP on the mitochondrial DNA. Applied Entomology and Zoology 37 : 437-446.

Nakahara S, Kato H, Kaneda M, Sugimoto T, Muraji M. 2001. Identification of Bactrocera dorsalis complex species (Diptara: Tephritidae) by PCR-RFLP analysis. II. A 175 study of genetic variation in B. dorsalis complex (Philippines population) and B. dorsalis (Taiwan population). Res. Bull. Plant Prot. Serv. Jpn. 37 : 69–73.

Nakahara S, Kato H, Kaneda M, Sugimoto T, Muraji M. 2002. Identification of the Bactrocera dorsalis complex (Diptera: Tephritidae) by PCR-RFLP analysis. III. Discrimination between B. philippinensis and B. occipitalis . Res. Bull. Plant Prot.Serv. Jpn. 38 : 73–80.

Nakahara S, Masaki M, Kaneda M, Sugimoto T, Muraji M. 2000. Identification of Bactrocera dorsalis complex species (Diptera: Tephritidae: Dacinae) by PCR-RFLP analysis. I. A study of variation in mitochondrial DNA D-loop region. Res. Bull. Plant Prot. Serv. Jpn. 36 :37–41.

Naeole CKM, Haymer DS. 2003. Use of oligonucleotide arrays for molecular taxonomic studies of closely related species in the oriental fruit fly (Bactrocera dorsalis) complex. Molecular Ecology Notes 3: 662-665.

Nishida R, Tan KH, Serit M, Lajis NH, Sukari AM, Takahashi S, Fukami H. 1988. Accumulation of phenylpropanoids in the rectal glands of male Oriental fruit fly, Dacus dorsalis . Experientia 44 : 534-536.

Nishida R, Tan KH, Fukami H. 1988. Cis-3.4-dimethoxycinnamyl alcohol from the rectal glands of male Oriental fruit fly, Dacus dorsalis . Chemical Express 3(4): 207-210.

Norrbom AL . 2004. Fruit fly (Tephritidae) host plant database: Version Nov., 2004. Last accessed 12 July, 2007, www.sel.barc.usda.gov:591/diptera/Tephritidae/TephIntro.html

Norrbom AL, Kim, K. 1988. A list of the reported host plants of the species of Anastrepha (Diptera: Tephritidae). USDA-APHIS-PPQ. (Publication 81-52). USDA APHIS Plant Protection and Quarantine, Beltsville, MD, USA.

Norrbom AL, Zucchi RA, Hernandez-Ortiz V. 1999. Phylogeny of the genera Anastrepha and Toxotrypana (Trypetinae: Toxotrypanini) based on morphology. pp. 299-342. In M. Aluja and A.L. Norrbom (eds.) Fruit flies (Tephritidae): phylogeny and evolution of behavior. Boca Raton, Florida, CRC Press, USA.

Oroño L, Paulin L, Alberti AC, Hilal M, Ovruski S, Vilardi JC, Rull J, Aluja M. Effect of Host Plant Chemistry on Genetic Differentiation and Reduction of Gene Flow Among Anastrepha fraterculus (Diptera: Tephritidae) Populations Exploiting Sympatric, Synchronic Hosts. Environ. Entomol. 42: 790-798.

Orr, H.A. Turfelli, M. 2001. The evolution of postzygotic isolation: accumulating Dobzhansky-Muller incompatibilities. Evolution , 55, 1085–1094.

Perkins MV, Fletcher MT, Kitching W, Drew RAI, Moore CJ. 1990. Chemical studies of rectal gland secretions of some species of Bactrocera dorsalis complex of fruit flies (Diptera: Tephritidae). J. Chem. Ecol. 16: 2475–2487.

Petit-Marty N, Vera MT, Calcagno G, Cladera JL, Segura DF, Allinghi A, Rodriguero M, Gómez Cendra P, Viscarret MM, Vilardi JC. 2004. Sexual behaviour and Mating 176

Compatibility of Four Populations of Anastrepha fraterculus (Diptera: Tephritidae) from Argentina. Ann. Entomol. Soc. Am. 97 (6): 1320-1327.

Petit-Marty N, Vera MT, Calcagno GE, Cladera JL, Vilardi JC. 2004. Lack of post- mating isolation between two population of Anastrepha fraterculus from different ecological regions in Argentina. En: libro editado de los trabajos completos presentados en el 6 th International Symposium on Fruit Flies of Economic Importance, pp 79-82.

Prezotto, L.F. 2008. Analise do ITS1 do DNA ribossomico em especies do complexo Anastrepha fraterculus . MSc thesis. Instituto de Biociências da Universidade de São Paulo.

Rocha LS, Selivon D. 2004. Studies of highly repetitive DNA in cryptic species of Anastrepha fraterculus (Diptera: Tephritidae) complex. In: B. Barnes (ed.) Proc. 6 th International Symposium on Fruit Flies of Economic Importance. Isteg Scientific Publications, Irene, South Africa, pp. 415-418.

Rull J, Abraham S, Kovaleski A, Segura DF, Islam A, Wornoayporn V, Dammalage T, Santo Tomas U, Vera MT. 2012. Random mating and reproductive compatibility among Argentinean and southern Brazilian populations of Anastrepha fraterculus (Diptera: Tephritidae). Bulletin of Entomological Research, doi:10.1017/S0007485312000016.

Rull J, Abraham S, Kovaleski A, Segura DF, Mendoza M, Liendo MC, Vera MT. 2013. Evolution of pre-zygotic and post-zygotic barriers to gene flow among three cryptic species within the Anastrepha fraterculus complex. Entomologia Experimentalis et Applicata 148: 213–222.

Salles LAB. 1995. Bioecologia e controle da mosca das frutas Sul Americana [Bioecology and control of South American fruit flies]. Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA), Centro de Pesquisa Agropecuária de Clima Temperado, Pelotas, Brazil. 58 pp.

San Jose M, Leblanc L, Geib SM, Rubinoff D. 2013. An evaluation of the species status of Bactrocera invadens (Drew, Tsuruta and White) and the Bactrocera dorsalis complex (Diptera: Tephritidae). Ann. Entomol. Soc. Amer. 106: 684–694.

Schluter D. 2009. Evidence for ecological speciation and its alternative. Science. 323(5915):737.

Schutze, M. K., A. Jessup, and A. R. Clarke. 2012a. Wing shape as a potential discriminator of morphologically similar pest taxa within the Bactrocera dorsalis species complex (Diptera: Tephritidae). Bulletin of Entomological Research 102:103-111.

Schutze, M. K., M. N. Krosch, K. F. Armstrong, T. A. Chapman, A. Englezou, A. Chomič, S. L. Cameron, D. L. Hailstones, and A. R. Clarke. 2012b. Population structure of Bactrocera dorsalis s.s., B. papayae and B. philippinensis (Diptera: Tephritidae) in southeast Asia: evidence for a single species hypothesis using mitochondrial DNA and wing- shape data. BMC Evolutionary Biology 12:130. doi:110.1186/1471-2148-1112-1130.

Schutze, M., A. Jessup, I. Ul-Haq, M. J. B. Vreysen, V. Wornoayporn, M. T. Vera, and A. R. Clarke. 2013. Mating compatibility among four pest members of the Bactrocera 177 dorsalis fruit fly species complex (Diptera: Tephritidae). Journal of Economic Entomology 106:695-707.

Segura, DF, Vera MT, Rull J, Wornoayporn V, Islam A, Robinson AS. 2011. Assortative mating among Anastrepha fraterculus (Diptera: Tephritidae) hybrids as a possible route to radiation of the fraterculus cryptic species. Biol. J. Linn. Soc. 102 : 346-354.

Segura DF, Vera MT, Wornoayporn V, Islam A, Robinson AS . Female choice and male lekking behaviour in Anastrepha fraterculus (Diptera: Tephritidae) hybrids from Argentina and Peru. Unpublished.

Selivon, D . 1996. Estudo sobre a diferenciação populacional em Anastrepha fraterculus (Wiedemann) (Diptera, Tephritidae). Doctoral thesis, Departamento de Biologia, Universidade de São Paulo, São Paulo, Brasil.

Selivon D, Perondini ALP. 1997. Evaluation of techniques for C and ASG banding of the mitotic chromosomes of Anastrepha species (Diptera, Tephritidae). Brazilian Journal of Genetics 20: 4–8.

Selivon, D., Morgante, J. S. & Perondini, A. L.P. 1997. Egg size, yolk masses extrusion and hatching behavior in two cryptic species of Anastrepha fraterculus . Brazilian Journal of Genetics 20 : 587-594.

Selivon D, Perondini ALP, Morgante JS. 1999. Haldane’s rule and other aspects of reproductive isolation observed in the Anastrepha fraterculus Complex (Diptera: Tephritidae). Genetics and Molecular Biology 22 :(4) 507-510.

Selivon D, Perondini ALP, Morgante JS. 2005. A genetic morphological characterization of two cryptic species of the Anastrepha fraterculus complex. (Diptera: Tephritidae). Annals of the Entomological Society of America 98: 367–381.

Severin HHP, Severin HC, Hartung WJ . 1914. The ravages, life history, weights of stages, natural enemies and methods of control of the Melon Fly ( Dacus cucurbitae Coq.). Annals of the Entomological Society of America VII (3): 178-207.

Shahjahan RM, Yesmin F. 2002. Polytene chromosome maps of the melon fly, Bactrocera cucurbitae (Coq.) (Diptera: Tephritidae). Genome. 15 (6): 1167-1174.

Smith PT, Kambhampati S, Armstrong KA. 2003. Phylogenetic relationships among Bactrocera species (Diptera: Tephritidae) inferred from mitochondrial DNA sequence. Molecular Phylogenetics and Evolution 26 : 8-17.

Smith-Caldas MRB, McPheron BA, Silva JG, Zucchi RA. 2001. Phylogenetic relationships among species of the fraterculus group ( Anastrepha : Diptera: Tephritidae) inferred from DNA sequences of mitochondrial cytochrome oxidase 1. Neotropical Entomology 30: 565–573.

Solferini VN , Morgante JS. 1987. Karyotype study of eight species of Anastrepha (Diptera, Tephritidae). Caryologia 40 : 229-241.

178

Steck GJ, Sheppard, WS. 1993. Mitochondrial DNA variation in Anastrepha fraterculus . In. M. Aluja & P. Liedo (eds), Fruit Flies, Biology and Management: 9-14. Springer-Verlag, New York. 492p.

Steck GJ. 1991. Biochemical systematics and population genetic structure of Anastrepha fraterculus and related species (Dipetera: Tephritidae). Ann. Entomol. Soc. Amer . 84 (1): 10- 28.

Steck GJ. 1999. Taxonomic status of Anastrepha fraterculus , pp. 13–20. In: The South American fruit fly, Anastrepha fraterculus (Wied.): advances in artificial rearing, taxonomic status and biological studies . IAEA-TECDOC-1064. Vienna: International Atomic Energy Agency, 202.

Stone A . 1942. The fruit flies of the genus Anastrepha . U S Dep. Agric. Misc. Publ. 439 : 1- 112.

Tan KH, Nishida R. 1996. Sex pheromone and mating competition after methyl eugenol consumption in the Bactrocera dorsalis complex. In: B. A. McPheron & G. J. Steck (eds.), Fruit Fly Pests – A World Assessment of their Biology and Management. St. Lucie Press, Delray, Florida, pp. 147–153.

Tan KH, Nishida R. 1998. Ecological significance of a male attractant in the defence and mating strategies of the fruit fly pest, Bactrocera papayae . Entomologia Experimentalis et Applicata 89 : 155-158.

Tan KH. 2000. Fruit Flies ( Bactrocera spp.) as pests and pollinators. Sabah Society Journal 17 : 37-56.

Tan KH. 2003a. Bactrocera dorsalis complex and its problem in control. Recent Trends Sterile Insect Technique Area-Wide Integrated Pest Management. Economic Feasibility Control Project, Farmer Organisation & Bactrocera dorsalis Complex Control Study. Okinawa, Japan, pp 103-112.

Tan KH. 2003b. Interbreeding and DNA analysis of sibling species within the Bactrocera dorsalis complex. Recent Trends Sterile Insect Technique Area-Wide Integrated Pest Management. Economic Feasibility Control Project, Farmer Organ. & Bactrocera dorsalis Complex Control Study. Okinawa, Japan, pp 113-122.

Tan, K. H. 2005. Interbreeding and DNA analysis of Bactrocera dorsalis sibling species. pp 113 – 122. In: Recent Trends on Sterile Insect Technique and Area-Wide Integrated Pest Management - Economic Feasibility, Control Projects, Farmer Organization and Bactrocera dorsalis Complex Control Study. Research Institute for the Subtropics, Okinawa, Japan.

Tan, K. H., I. Tokushima, H. Ono, and R. Nishida. 2011. Comparison of phenylpropanoid volatiles in male rectal and pheromone gland after methyl eugenol consumptions, and molecular phylogenetic relationship of four global pest fruit fly species: Bactrocera invadens , B. dorsalis , B. correcta and B. zonata . Chemoecology 21:25-33.

Tan, K. H., S.L. Wee, H. Ono, and R. Nishida. 2013. Comparison of methyl eugenol metabolites, mitochondrial COI, and rDNA sequences of Bactrocera philippinensis (Diptera: 179

Tephritidae) with those of three other major pest species within the dorsalis complex. Applied Entomology and Zoology 48:275-282.

Vanícková L., A. Svatoš, J. Kroiss, M. Kaltenpoth, R. Rufino Do Nascimento, M. Hoskovec, R. Brízová, and B Kalinová. 2012. Cuticular Hydrocarbons of the South American Fruit Fly Anastrepha fraterculus : Variability with Sex and Age. J. Chem Ecol.

Vera MT, Cáceres C, Wornoayporn V, Islam A, Robinson AS, de la Vega MH, Hendrichs J, Cayol JP. 2006. Mating Incompatibility among Populations of the South American Fruit Fly Anastrepha fraterculus (Wied.) (Diptera: Tephritidae). Ann. Entomol. Soc. Am . 99 : 387-397.

Virgilio M, Backeljau T, De Meyer M. 2007a. FAR complex and barcoding. 2nd International Barcode of Life Conference (Taipei, Taiwan) : 142.

Virgilio M, Backeljau T, De Meyer M. 2007b. Incongruence of phylogenetic signals and shared polymorphisms prevent the molecular characterization of the Ceratitis fasciventris , C. anonae , C. rosa complex (Diptera: Tephritidae)”, abstract of 11th Congress of the European Society for Evolutionary Biology (Upsalla, Sweden) : 520.

Virgilio M, Backeljau T, Barr N, De Meyer M. 2008. Molecular evaluation of nominal species in the Ceratitis fasciventris , C. anonae , C. rosa complex (Diptera: Tephritidae). Molecular Phylogenetics and Evolution 48, 270-280.

Virgilio M, Delatte H, Backeljau T, De Meyer M. 2010. Macrogeographic population structuring in the cosmopolitan agricultural pest Bactrocera cucurbitae (Diptera: Tephritidae). Molecular Ecology 19, 2713–2724.

Virgilio M, Delatte H, Quilicy S, Backeljau T, De Meyer M. 2013. Cryptic diversity and gene flow among three African agricultural pests: Ceratitis rosa, Ceratitis fasciventris and Ceratitis anonae, (Diptera, Tephritidae). Molecular Ecology (2013) doi: 10.1111/mec.12278.

Wang Y, Yu H, Raphael K, Gilchrist AS. 2003. Genetic delineation of sibling species of the pest fruit fly Bactocera (Diptera: Tephritidae) using microsatellites. Bull. Entomol. Res. 93 : 351–60

Waterhouse DF. 1993. Biological Control: Pacific Prospects - Supplement 2. ACIAR Monograph No. 20, viii, 138pp.

Wee SL, Tan, KH. 2000. Sexual maturity and intraspecific mating success of two sibling species of the Bactrocera dorsalis complex. Entomologia Experimentalis et Applicata 94 : 133-139.

Wee SL, Hee AKW, Tan KH. 2002. Comparative sensitivity to and consumption of methyl eugenol in three Bactrocera dorsalis complex sibling species. Chemoecology 12 : 29-33.

Wee SL, Tan KH. 2005. Evidence of natural hybridization between two sympatric sibling species of Bactrocera dorsalis complex based on pheromone analysis. Journal of Chemical Ecology 31 (4): 845-858.

180

Wee SL, Tan KH. 2007. Temporal accumulation of phenylpropanoids in male fruit flies, Bactrocera dorsalis and B. carambolae (Diptera: Tephritidae) following methyl eugenol consumption. Chemoecology 12 : 193-197.

Wee SL, Tan KH, Nishida R. 2007. Pharmacophagy of methyl eugenol by males enhances sexual selection of Bactrocera carambolae (Diptera: Tephritidae). Journal of Chemical Ecology 33 : 1272-1282.

White IM, Elson-Harris MM. 1992. Fruit flies of economic significance: their identification and bionomics. CAB International, Wallingford, Oxon, UK and The Australian Center for Agricultural Research, Canberra, Australia. 601 p.

White IM, Hancock DL. 1997. Indo-Australasian Dacini fruit flies. (Computer Aided Biological Identification Key). International Institute of Entomology, London. Compact Disk.

Yamada S, Selivon D. 2001. rose, an eye color mutation in a species of the Anastrepha fraterculus complex (Diptera, Tephritidae). Ann. Entomol. Soc. Am. 94: 592-595.

Yasmin F. 2013. Chromosomes of Bactrocera papaya Drew & Hancock and Bactrocera carambolae Drew & Hancock (Diptera: Tephritidae): Mitotic Metaphase Karyotype and Polytene Genome. PhD Thesis, Malaysia.

Yong HS. 1994. Hydroxybutyrate dehydrogenase electromorph distinguishes Bactrocera dorsalis (Insecta: Diptera: Tephritidae) from sibling taxa in Peninsular Malaysia. In: Yong HS, Khoo SG, eds. Current research on tropical fruit flies and their management . Kuala Lumpur: The Working Group on Malaysian Fruit Flies, pp 23–26.

Yong HS. 1995. Genetic differentiation and relationships in five taxa of the Bactrocera dorsalis complex (Insecta: Diptera: Tephritidae). Bull. Entomol. Res. 85 : 431–435

Zacharopoulou A. 1987. Cytogenetic analysis of mitotic and salivary gland chromosomes in the medfly Ceratitis capitata . Genome 29: 67–71.

Zacharopoulou A, Augustinos AA, Sayed WAA, Robinson AS, Franz G. 2011. Mitotic and polytene chromosomes analysis of the oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae). Genetica, 139: 79-90.

Zacharopoulou A, AA Sayed WAA, Augustinos AA, Yesmin F, Robinson AS, Franz G. 2011. Analysis of mitotic and polytene chromosomes and photographic polytene chromosome maps in Bactrocera cucurbitae (Diptera: Tephritidae). Annals of the Entomological Society of America, 104 (2): 306-318.

Zimowska GJ, Handler AM. 2005. piggyBac transposons in Bactrocera dorsalis and their use for species identification. Eight Exotic Fruit Fly Symposium, California, March, 2005. Abstract.