Use of Symbiotic Bacteria to Reduce Mass-Rearing Costs and Increase Mating Success in Selected Fruit Pests in Support of SIT Application

IAEA-D41024-CR-4 LIMITED DISTRIBUTION WORKING MATERIAL

Report of the Fourth Research Coordination Meeting of an FAO/IAEA Coordinated Research Project, held in Vienna, Austria, from 17 to 21 May 2017

Reproduced by the IAEA Vienna, Austria 2017

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TABLE OF CONTENTS BACKGROUND ...... 3 CO-ORDINATED RESEARCH PROJECT (CRP) ...... 8 FOURTH RESEARCH CO-ORDINATION MEETING (RCM) ...... 8 1 LARVAL DIETS AND RADIATION EFFECTS ...... 10 BACKGROUND SITUATION ANALYSIS ...... 10 INDIVIDUAL PLANS ...... 13 1.1. Cost and quality of larval diet ...... 13 1.2. Preventing the growth of deleterious microorganisms ...... 23 1.3. Improving mass rearing efficiency and quality of the insects produced ...... 27 2 PROBIOTICS ...... 35 BACKGROUND ...... 35 3 SYMBIONTS AND NOVEL CONTROL TOOLS ...... 73 LOGICAL FRAMEWORK ...... 120 LIST OF REFERENCES ...... 127 ANNEX 1 (LIST OF PARTICIPANTS) ...... 134 ANNEX 2 (AGENDA) ...... 137 ANNEX 3 (WORKING GROUPS) ...... 141 ANNEX 4 (ABSTRACTS OF PRESENTATIONS) ...... 142

BACKGROUND

Flies and moth pests cause significant and widespread damage to fruit and vegetable production. Insecticide application remains the predominant method of controlling these pests. Indeed, these high value crops are the main targets of agrochemicals, receiving currently 32% of all applications in agriculture. Despite this growing dependence on agrochemicals, suppression of the pest populations is frequently inadequate. In addition, due to regulation, pest resistance, environmental and human health concerns, there is an increasing demand for the replacement of the intensive use of these chemicals by environmentally friendly, effective and sustainable methods, within integrated management approaches. Chief among these is the sterile insect technique (SIT) and related biological control applications.

The efficacy of the SIT is determined by the quality of insects mass-reared in production facilities, irradiated and released in the field. Constraints on the quality of these insects are manifest at every stage of production and major efforts have been made to assure quality within reasonable economic limits. Indeed, the increased use of the SIT is frequently limited by cost-benefit considerations, as the mass-rearing of target insects, their delivery and release in prime condition may be in some situations prohibitively expensive. Accordingly, efforts to streamline the SIT process, combining improved quality of sterile insects with reduced production costs should enable the increased application of this approach.

In the past, efforts to improve sterile insect quality for fruit fly pests have focused on colonization, mass-rearing, quality control and pre-release handling. In the current proposal, we seek to extend these approaches to manipulating the diverse microorganisms associated with the fruit pests targeted in SIT operations to protect fruit and vegetable crops.

Insects are indisputably the dominant multicellular organisms in terrestrial habitats. As such they maintain intricate and complex interactions with other organisms in their habitat. Some of these interactions, such as those between insects and plants, or insects and vertebrates, have been extensively studied. On the other hand, the associations between insects and microorganisms, while pervasive and of paramount ecological and evolutionary importance, are only gradually being understood. Insects depend on symbiotic associations with a variety of microorganisms, which affect many aspects of host biology and physiology including nutrition, immunity, mating behaviour and reproduction.

The insect symbiotic associations are currently artificially divided into four categories: The first category includes symbionts that provide nutrients such as amino acids and vitamins to their hosts through mutualistic associations. The second category includes symbionts that provide their hosts with the ability to survive heat stress, to develop resistance to parasitic wasps and/or microbial pathogens, and to exhibit altered host plant preference. The third

category includes symbionts that manipulate the reproductive properties of their hosts, inducing phenomena such as parthenogenesis, feminization, male-killing and cytoplasmic incompatibility (CI), which is a kind of male sterility. The fourth category includes symbionts that help insects to detoxify xenobiotics such as pesticides, plant related defensive molecules.

Symbiotic microorganisms and the SIT Symbiotic organisms can be important at all stages of the SIT. We seek to resolve four key questions related to them: 1. Can symbionts help reduce the cost of production and increase mass-reared sterile insect quality? 2. How are symbiotic associations affected by radiation and can they be ameliorated? 3. Can they be used as probiotics during the pre-release period to improve sterile insect quality? 4. Can they be used to develop novel pest control tools, complementary to the SIT? These questions are expanded in the following paragraphs.

1. Costs of production and increased quality Under natural conditions, fruits used by larvae have extremely low amino acid contents. Ovipositing females inject their eggs into fruit along with bacteria that fix atmospheric nitrogen and others that break down the fruit to produce nutrients essential for larval growth. The mass-rearing process frequently disassociates the reared insects from their native microflora, allowing the proliferation in larval media of opportunistic microorganisms that may not be beneficial. Yeasts to provide nourishment, and chemicals to suppress opportunistic microorganisms, represent by far the largest cost of larval diets. Adding endogenous symbiotic bacteria to the artificial larval and adult diet may significantly:  reduce mass-rearing costs by eliminating the need for yeasts and chemicals  prevent the growth of deleterious microorganisms  improve mass-rearing efficiency and quality of the insects produced

2. Effects of radiation Evidence suggests that radiation of mass-reared flies can disrupt symbiotic associations by favouring some bacterial species and suppressing others. Understanding the effects of radiation may enable us to design responses that address them in a manner that optimizes the SIT efficiency.

3. Probiotics In nature, symbiotic bacteria become established in the gut of adult flies. These appear to play an important role in the reproductive success of males. The complement of bacteria present in released males following mass-rearing and irradiation may differ from their wild counterparts enough to impede their performance. There is preliminary evidence that restoring the symbiotic bacteria, prior to release, can significantly improve their sexual performance. Enriching the sterile insect diet of the Mediterranean fruit fly with the naturally occurring bacterium, Klebsiella oxytoca, significantly improved sterile male mating competitiveness in the laboratory and in field cages. In addition, bacterially enriched sterile males inhibited female receptivity to re-mating more efficiently than sugar fed males and survived longer periods of starvation. These results suggest that restoring key bacteria to mass-reared sterile flies prior to their release is a valid approach to improve the efficacy of the SIT. It is

worthwhile to validate this approach at an operational level and to extend it to other insects targeted by the SIT.

4. Symbiotic organisms and novel control tools Certain symbiotic bacteria are known to manipulate mating behaviour and reproduction of their hosts. Over the last 18 months, in the frameworks of the current Coordinated Research Project, Wolbachia as well as other microorganisms that affect the reproduction of tephritids have been characterized. Wolbachia infection was recorded in Anastrepha striata, A. serpentina, A. obliqua and A. fraterculus. Other studies have demonstrated the occurrence of Wolbachia in additional Anastrepha species of minor economic importance. Regarding Bactrocera species, Wolbachia has been detected in B. dorsalis, B. cucurbitae, B. zonata, B. correcta, B. tryoni and B. tau. Detailed characterization of Wolbachia presence in these species should be conducted. A thorough screening of medfly populations from Brazil, Turkey and Argentina revealed no Wolbachia infection on the sampled populations. Besides Wolbachia the occurrence of other symbionts, such as Cardinium, Arsenophonus, Spiroplasma, have been screened in Bactrocera spp. and C. capitata; however, confirmation of their occurrence is pending.

Incompatible insect technique (IIT) employs cytoplasmic incompatibility, induced by insect symbionts such as Wolbachia species. In a Wolbachia-based IIT strategy, female sterility is artificially sustained by repeated releases of cytoplasmically incompatible males. Since Wolbachia is not paternally transmitted, the infection type present in the release strain does not become established in the field. Similar to the SIT, the increasing ratio of incompatible matings over time can lead to population suppression. This strategy has been successfully tested under laboratory conditions for two major agricultural pests, the Mediterranean fruit fly and the olive fly. It is worthwhile for such an approach to be validated and extended, alone and/or in conjunction with the SIT, to other target insect pest species. Field data from Brazil, Turkey and Argentina demonstrating no Wolbachia infection in wild medfly populations suggest that medfly is a good candidate for the application of IIT. Considering that wild populations of A. ludens are not infected with Wolbachia, current efforts have focused on evaluating the possibility of IIT for this species. First steps included establishing transinfected mass reared A. ludens lines with Wolbachia strain using A. striata as donor. Possible application of IIT to control Bactrocera species (based on the current characterization of Wolbachia prevalence in different Bactrocera species) should be explored. Besides IIT other possible applications of the Wolbachia-tephritid association for controlling fruit flies should be explored.

In conclusion, symbiosis, a powerful new field, increases our understanding of basic biological questions, and can have profound effects on applied fields, from medicine to agriculture. This CRP aims at the characterization and harnessing of endogenous symbiotic communities in order to: (a) reduce costs of mass-rearing, (b) determine the effects of radiation, (c) use symbionts as probiotics and (d) develop novel and SIT-compatible insect

control tools. We believe that this initiative will lead to better and more cost-effective SIT programmes against fruit insect pests.

CO-ORDINATED RESEARCH PROJECT (CRP)

This Coordinated Research Project (CRP) is based on a Consultants Meeting that was held from 7-11 February 2011 in Vienna, Austria (report available) to assess the potential for conducting co-ordinated R&D in larval and adult insect for releases, and to formulate a proposal for a CRP on Use of Symbiotic Bacteria to Reduce Mass-Rearing Costs and Increase Mating Success in Selected Fruit Pests in Support of SIT Application.

The overall objective of this CRP D4.10.24, approved for the period 2012-2017, is to ultimately reduce the cost and increase the effectiveness of SIT programmes by reducing costs of mass-rearing, determining the effects of radiation, using symbionts as probiotics and developing novel and SIT-compatible insect control tools.

FOURTH RESEARCH CO-ORDINATION MEETING (RCM)

Ninegteen scientists from 16 countries attended the second RCM, held in Vienna, Austria from 17-21 May, 2017. The list of participants, which included CRP contract and agreement holders, as well as 10 additional observers, is given in Annex 1. The agenda for the meeting is attached in Annex 2.

During the first two days of the meeting RCM participants presented research relevant to the CRP, as well as to present the resrch that was carried-out during the length of the CRP.

During the last three days of the meeting, general discussions were held to define and review the thematic areas of the CRP (Table 1), the review of the general and specific R&D objectives for the 5 years of the CRP (Sections 1, 2, and 3), and outputs achieved at the end of the CRP. Furthermore, participants were divided into three working groups (Annex 3) to develop more detailed report for the outcomes achived during the the entere length of the CRP. During the las session a discussions was held on the final papers for the special issue in the BMC Microbiology.

Abstracts of the presentations are presented in Annex 4 and a copy of all PowerPoint presentations was made available to all participants at the end of the RCM.

Table 1. Thematic areas in relation to pest species being addressed by researchers (for main areas of work, names appear in bold).

1. Larval diets and 2. Probiotics (adult 3. Symbionts and Species radiation effects diets) novel control tools Anastrepha A. fraterculus Segura (ARG) Segura (ARG)

A. ludens Melgar (GUA) Melgar (GUA) Mateos (USA) Schuenzel (USA)

A. obliqua, Hernandez (MEX) Mateos (USA) A. serpentina, A. striata

Bactrocera B. cucurbitae Khan (BGD) Khan (BGD) Khan (BGD) Hire (IND) Hire (IND) Hire (IND)

Dyall (MAR) Tsiamis (GRE)

B. dorsalis complex Khan (BGD) Khan (BGD) Niu (CPR) Niu (CPR) Tsiamis (GRE) Niu (CPR) Hire (IND) Bourtzis (IAEA) Khan (BGD)

B. minax Niu (CPR) Niu (CPR) Niu (CPR)

B. oleae Yuval (ISR) Sacchetti (ITA) Yuval (ISR)

B. tryoni Reynolds (AUS) Reynolds (AUS)

B. zonata Dyall (MAR) Hire (IND) Khan (BGD) Tsiamis (GRE) B. correcta Hire (IND) Khan (BGD) Tsiamis (GRE) Ceratitis C. capitata Papadopoulos (GRE) Papadopoulos (GRE) Papadopoulos (GRE) Melgar (GUA) Melgar (GUA) Tuncbilek (TUR) Garcia (SPA) Garcia (SPA) Tuncbilek (TUR) Yañez (CHI)

Dirioxa D. pornia Crisp (AUS)

1 LARVAL DIETS AND RADIATION EFFECTS

Background Situation Analysis Under natural conditions, fruits used by larvae have extremely low amino acid contents. Ovipositing females inject their eggs into fruit along with bacteria that most probably fix atmospheric nitrogen and others that break down the fruit to produce nutrients essential for larval growth. The mass-rearing process frequently disassociates the reared insects from their native microflora, allowing the proliferation in larval media of opportunistic microorganisms that may not be beneficial. Yeasts to provide nourishment, and chemicals to suppress opportunistic microorganisms, represent by far the largest cost of larval diets. Adding endogenous symbiotic bacteria to the artificial larval diet may significantly:

A. reduce mass-rearing costs by decreasing, replacing or eliminating the need for yeasts and chemicals B. prevent the growth of competing microorganisms C. improve mass-rearing efficiency and quality of the insects produced

Subtheme A: Cost and quality of larval diet

Current knowledge:

A1. General Currently all mass rearing facilities of different fruit fly pest species use artificial larval diet which consists of bulking agents, yeasts, sugars, preservatives, acidifying agents and water. The ingredient that mainly affects the cost (other than the labour cost) and the quality of the larval diet is yeast. Yeast supplements can make up to 50% of the total larval diet cost. The presence of deleterious microorganisms is also an important issue and is mainly related to the type and the availability of bulking agents at the different facilities location.

A2. Species level background Artificial larval diet is available for the following species: Ceratitis capitata (medfly), Bactrocera oleae (olive fly), B. dorsalis (oriental fruit fly), Zeugodacus cucurbitae (melon fly), B. tryoni (Queensland fruit fly), B. zonata, B. tau, Anastrepha obliqua, A. ludens, A. fraterculus, A. serpentina, A. striata, and Dirioxa pornia. Ceratitis capitata, Anastrepha species, Bactrocera species (other than B. oleae), and D. pornia: all facilities use a diet of a very similar composition, the difference mainly being to the source of the bulking agents, the protein source (different species of yeast), and the pH level which ranges from 3.5 to 4.5. Novel diets (liquid and gel diet) are under evaluation and may facilitate the incorporation of beneficial microorganisms. Bactrocera oleae: Being a monophagous pest species on a fruit of an uncommon composition, olive fly larval diet requires specific ingredients including soy hydrolysate, which accounts for more than 50% of the total cost, and cellulose as bulking agent. The diet requires further quality improvement and cost reduction.

Gaps identified: A1. General: 1. Replacement of the bulking agents 2. Replacement (reduction) of protein sources 3. Optimizing the use of preservatives 4. Egg/larval inoculation with beneficial microbes 5. Different facilities have access to different larval diet ingredients

A2. Species level background: Each one of the five gaps identified needs to be considered for all fruit fly pest species targeted in this CRP: Ceratitis capitata, Bactrocera oleae, Z. cucurbitae, B. tryoni, B. dorsalis, B. zonata, B. tau, B. correcta, Anastrepha obliqua, A. ludens, A. fraterculus, A. serpentina, A. striata, A. grandis, and Dirioxa pornia. Particular emphasis should be given to B. oleae being the species with the most inefficient larval diet. The need of a specialized scientist with good knowledge of food industry (animal nutrition) is recognized.

Subtheme B: Preventing the growth of deleterious microorganisms

Current knowledge:

B1. General: The presence of deleterious microorganisms is a common problem for all mass rearing facilities. A monitoring protocol is in use in some facilities and is based on classical microbiological tests (API tests). Using this protocol, bacteria like Lactobacillus spp, Pseudomonas spp., Morganella spp., Serratia spp., Raoultella sp., Klebsiella sp. and yeasts have been identified. However, these tests have limited potential to identify microorganisms at the species level. Therefore, molecular approaches need to be incorporated. In addition, there is an urgent need to develop a monitoring protocol for the detection of deleterious microorganisms in all mass rearing facilities at three levels: (a) the ingredients, (b) the larval diet and (c) the environment (rearing equipment, air conditioning, HEPA filters, etc.).

B2. Species level background: The currently available monitoring protocol is only in use in the facilities of El Pino and Petapa, MoscaMed-USDA [Guatemala], MoscaFrut, MoscaMed [Mexico], Valencia [Spain], Mission Texas [USA] which rear the followin species: Ceratitis capitata, Anastrepha obliqua, and A. ludens.

Gaps identified:

B1. General: 1. There is an urgent need to combine microbiological and molecular methods for the accurate identification of deleterious microorganisms. Isolation and characterization of bacterial strains will also be required. 16S rRNA characterization of the deleterious microorganisms is inadequate. Other genetic markers, and/or biochemical methods, and genomic data of the pathogenic strains are required for characterization and typing at species and strain level. Revision of existing MLST protocols and development of new protocols are needed.

2. Uncover the conditions (pH, aeration, water activity, ingredients) that are conducive to the development of deleterious microorganisms in the larval diet, the rearing rooms and equipment. 3. Cost-effective preservatives to be tested in view of a complete list of deleterious microorganisms (see gap 1). 4. Beneficial microorganisms to be used as competitors of deleterious bacterial species (once gaps 1 and 3 are filled). 5. A revised monitoring protocol should be developed and implemented upon the filling of the above gaps. 6. An e-platform should be developed to integrate all data collected (particularly from gaps 1 and 2).

B2. Species level background: All the above gaps need to be addressed for each one of the following species: Ceratitis capitata, Bactrocera oleae, B. cucurbitae, B. tryoni, B. dorsalis, B. zonata, Anastrepha obliqua, A. ludens, A. fraterculus, A. serpentina, A. striata, and Dirioxa pornia.

Subtheme C: Improving mass rearing efficiency and quality of the insects produced

Current knowledge:

C1. General: Despite the great progress achieved during the last 40 years in the mass rearing protocols of major agricultural fruit fly pests, there is still plenty of room for improving the efficiency as well as the quality of the insects produced. Recent advancements in the field of insect symbiosis suggest the presence of a diverse group of symbiotic microorganisms which play a major role on the biology and the physiology of insects. Therefore, a deep knowledge on the composition, the dynamics and the functional role of insect symbiotic communities is required and this will certainly enhance the production of higher quality mass reared insects.

C2. Species level background: The species currently under artificial rearing can be divided into three groups depending on the efficiency and the quality of insects produced: Ceratitis capitata, A. ludens, A. obliqua are the only species on the top of this list of the most efficient rearing protocols; B. oleae is in the bottom while all the others are in the middle.

Gaps identified:

C1. General: 1. Monitoring (dynamics and functional role) of the insect-associated and other microorganisms reared in different artificial larval diets by microbiological and or molecular approaches. 2. Monitoring (identification, dynamics and functional role) of the symbionts associated with the fruit flies (larvae) under natural conditions (different host plants, geographic regions, seasons, etc.). 3. Define the beneficial microorganisms based on the outcome of (1) and (2). 4. Mass production of beneficial microorganisms in large scale should be provided in the early stages of the insect development (fermentors).

5. Develop robust and unified microbial preservation protocols. 6. Develop robust and efficient inoculation methods for the beneficial microorganisms. 7. Technical and economic feasibility for the production and the inoculation of beneficial microorganisms. 8. Gaps 4-6 can be addressed once cultivation protocols for insect beneficial microorganisms become available or developed.

C2. Species level background: Intensive research efforts to characterize the symbiotic communities of several fruit fly pest species (for example, Ceratitis capitata, Bactrocera oleae, B. cucurbitae, B. dorsalis, B. minax, B. zonata, Anastrepha obliqua, A. fraterculus, A. ludens, A. serpentina, A. striata, B. tryoni, and D. pornia). Others remain to be characterized.

Individual plans

1.1. Cost and quality of larval diet

Species: Ceratitis capitata Participants: Hernán Donoso, Carolina Yáñez (Chile)

5 years plan  Identification of microbial communities in larvae and pupae of the laboratory colony (under mass rearing and small scale conditions) by molecular (DGGE, 16S rRNA gene sequencing) and classical culture-dependent approaches.  Identify suitable beneficial microorganisms for probiotic applications at larval stage.  QC field tests using standard field cage in order to assess the real impact of the provision of probiotics during the period prior to their release, with special attention to the eventual increase of competitiveness, dispersal and survival.

Achievements at the end of the CRP

 Bacterial strains from larvae and pupae of the mass-rearing facility (injection and release streams) were isolated and identified.  Bacterial communities from larvae and pupae of the mass-rearing facility (injection and release streams) were characterized using DGGE.

Gaps  Identification of suitable beneficial microorganisms for probiotic applications.  Development of protocols for addition of bacteria to larval diet (laboratory and mass- rearing scale).  Quality assessment of the bacterial fed larvae.

Recommendations  Unify protocols for addition of bacteria to larval diet and evaluation of bacterial fed larvae in all mass-reared facilities.  Before introducing bacteria to diets, genomic analysis should be done in order to determine if pathogenicity exists.

Participants: Dina Melgar, Pedro Rendón, Felipe Jerónimo (Guatemala)

5 years plan  Identification of microbial communities in egg, larvae and pupae of wild and mass reared medfly, wild infested fruits and mass rearing larval diet by molecular and classical approaches (DGGE, 16S rRNA sequencing).  Identify suitable beneficial microorganisms for probiotic applications at larval stage.  Development of a formulation for probiotic applications at mature egg and/or the first/second larvae instars.

Achievements at the end of the CRP  Microbial profile characterization in egg suspension by biochemical test  Characterization of beneficial bacterial (Klebsiella sp., Enterobacter sp. and Raoultella sp.  Protocol to introduce probiotics at mass rearing facility.  Evaluation of addition of Klebsiella oxytoca in the egg´s suspension. Gaps  Identification of microbial communities in egg, larvae and pupae of wild and mass reared medfly (different strains and colonies), wild infested fruits and mass rearing larval diet by molecular (DGGE, 16S rRNA sequencing) and classical approaches has been not completed.

Participants: Jaime García de Oteyza, Teresa Navarro (Spain)

5 years plan  Identification of microbial communities in egg, larvae and pupae of wild and mass reared medfly (different strains and colonies), wild infested fruits and mass rearing larval diet by molecular and classical approaches (DGGE, 16S rRNA sequencing).  Identify suitable beneficial microorganisms for probiotic applications at larval stage.  Development of a formulation for probiotic applications at mature egg and/or the first/second larvae instars.

Achievements at the end of the CRP  Bacterial strain identification for all isolates has been completed (Klebsiella sp., Raoultella sp.).  DNA profiles for pure cultures have been produced.  16S rRNA amplicon has been produced for the characterization of the gut microbial communities.  Characterization of microbial communities in egg, larvae and pupae of wild and mass reared medfly (different strains and colonies), wild infested fruits (from different seasons and regions) and mass rearing larval diet by molecular (DGGE, 16S rRNA gene sequencing) and classical approaches has been completed.  Protocol for probiotic application at first larval instar has been developed.  Identification of yeasts in wild and mass reared medfly adults has been carried out by molecular approaches (ITS).  Strain collection (medfly isolates) available.

Gaps  Develop protocols for identification of bacterial strains at species level, and for evaluating performance as probiotics.

Species: Anastrepha ludens Participants: Dina Melgar (Guatemala)

5 years plan  Identification of microbial communities in egg, larvae and pupae of wild and mass reared medfly (different strains and colonies), wild infested fruits and mass rearing larval diet by molecular (DGGE, 16S rRNA sequencing) and classical approaches.  Identify suitable beneficial microorganisms for probiotic applications at larval stage.  Development of a formulation for probiotic applications to mature eggs.

Achievements at the end of the CRP  Microbial profile characterization in egg suspension by biochemical tests.  Beneficial bacteria characterization (Klebsiella sp., Enterobacter sp. and Raoultella sp.  Protocol to introduce probiotics at mass rearing facility.  Evaluation of addition of Raoultella ornithinolitica in the egg´s suspension.

Gaps  Identification of microbial communities in egg, larvae and pupae of wild and mass reared medfly, wild infested fruits and mass rearing larval diet by molecular (DGGE, 16S rRNA sequencing) and classical approaches has been not completed.

Participants: Emilio Hernández, Pablo Liedo (México)

5 years plan  Identification of microbial communities in wild and laboratory larvae (third instar) by using molecular and classical microbiological phenotyping approaches.  Identify suitable beneficial microorganisms for probiotic applications at larval stage.  Development of protocols for probiotic introduction into larval diet.

Achivements at the end of the CRP  Identification of microbial communities from wild and laboratory larvae (third instar) and adult populations using molecular approaches (pyrosequencing) has been finalized but not published.

Recommendations  Continue with the isolation of the cultivable bacteria.

Participants: Erin Schuenzel, Bacilio Salas, Hugh Conway, Don Vacek (USA)

5 years plan

 Identification of microbial communities in mass-reared eggs and third instar-larvae by using molecular and classical microbiological phenotyping approaches.  Identify suitable beneficial microorganisms for probiotic applications at larval stage.

Achievements at the end of the CRP  650 isolates were identified using molecular techniques to bacterial genus or species using 16S rDNA and fungal genus and species using ITS.  No beneficial isolates were identified using small cup test of larval survivorship for over 400 of the identified isolates. Gaps  Culture independent methods (NGS) needs to be completed for the eggs, larvae (1st, 2nd and 3rd larval instar), pupa and environmental surfaces at mass-rearing facility. Recommendations  Perform culture-independent surveys to compare to culture-dependent isolates.  Perform more small-cup tests on any otu not previously tested if cultured organism is available. Species: Anastrepha obliqua Participants: Emilio Hernández, Pablo Liedo (México)

5 years plan  Identification of microbial communities in wild and laboratory third instar-larvae by using molecular and classical microbiological phenotyping approaches.

 Identify suitable beneficial microorganisms for probiotic applications at larval stage.

 Development of protocols for probiotic introduction into larval diet. Achivements at the end of the CRP  Identification of microbial communities from wild and laboratory larvae (third instar) and adult populations using molecular approaches (pyrosequencing) has been finalized but not published.

Gaps  The results of the indetification should be analysed and published.

Recommendations  Finalize the identification and publish the results.  Continue with the isolation of the cultivable bacteria

Species: Anastrepha serpentina Participants: Emilio Hernández, Pablo Liedo (México)

5 years plan

 Identification of microbial communities in wild and laboratory third instar-larvae by using molecular and classical microbiological phenotyping approaches.

 Identify suitable beneficial microorganisms for probiotic applications at larval stage.

Gaps  The results of the indetification should be analysed and published.

Recommendations  Finalize the identification and publish the results.  Continue with the isolation of the cultivable bacteria.

Species: Anastrepha striata Participants: Emilio Hernández, Pablo Liedo (México)

5 years plan  Identification of microbial communities in wild and laboratory third instar-larvae by using molecular and classical microbiological phenotyping approaches.

 Identify suitable beneficial microorganisms for probiotic applications at larval stage.

Gaps  The results of the indetification should be analysed and published.

Recommendations  Finalize the identification and publish the results.

Species: Anastrepha fraterculus Participants: Fernando Consoli (Brazil)

5 years plan  Identification of microbial communities in larvae and adults of wild and lab reared fruit flies (different strains and colonies) by molecular approaches (16S rDNA library and/or 16S rDNA pyrosequencing).  Identification of the culturable microbiota of larvae and adult of a lab colony.

 Identify suitable beneficial microorganisms for probiotic applications at larval stage. Achivements at the end of the CRP  Culturable microbiota associated with the larval and adult gut of a lab strain reared on papaya has been completed.  Identification of microbial communities in larvae and adult of wild and lab reared fruit flies by molecular approaches (16S rDNA library) has been completed.

Species: Zeugodacus cucurbitae Participants: Mahfuza Khan (Bangladesh) Collaborators: Kostas Bourtzis (FAO/IAEA), and George Tsiamis (Greece)

5 years plan  Identification of gut microbial communities of larvae of laboratory reared (host and artificial larval diet), and artificial larval diets by classical microbiological/biochemical approaches and molecular techniques (16S rDNA amplicon sequencing).  Identify suitable beneficial microorganisms for probiotic applications at larval stage.  Assess the quality parameters of Z. cucurbitae reared from bacteria incorporated larval diets.

Achievements at the end of CRP  The gut microbial communities were determined for eggs, the midgut of third instar larvae, and adults of laboratory (host-sweetgourd) reared Z. cucurbitae using classical microbiological/biochemical tests, and API kits.  Establishment of larval rearing of Z. cucurbitae on liquid diet based on the formulation of Dr. Stella Chang (USDA).  The gut microflora has been determined from a lab colony using amplicon sequencing. Proteobacteria is the dominant Phylum. Four developmental stages were examined (larva, 1-day, 5-days, and 15-days old adults).

Gaps  Although the gut bacterial community were identified for laboratory reared flies, the bacterial community of wild flies is not known.  The use of liquid larval diet for large scale mass rearing of Z. cucurbitae was not found to be feasible.

Recommendations  Future endeavour should be made on the development of appropriate larval diets for mass rearing of Z. cucurbitae.  Selected gut bacterial isolates can be tried as probiotic to improve the quality parameters of reared flies.

Species: Bactrocera dorsalis complex Participants: Mahfuza Khan (Bangladesh) Collaborators: Kostas Bourtzis (IAEA), and George Tsiamis (Greece)

5 years plan

 Identification of gut microbial communities of larvae of laboratory reared (host and artificial larval diet), by classical microbiological/biochemical approaches and molecular techniques (16S rDNA amplicon sequencing).  Identify suitable beneficial microorganisms for probiotic applications at larval stage.  Assess the quality parameters of B. dorsalis reared from bacteria incorporated in liquid and gel larval diets.

Achievements at the end of CRP  The gut microbial communities were identitfed for egg, the mid gut of third instar larvae, and adults of laboratory (host-banana and liquid larval diet) reared B. dorsalis by classical microbiological/biochemical tests, and API kits. The identity of selected gut bacterial species of third instar larvae was confirmed by 16S rRNA analysis.  Assessment on the quality of B. dorsalis developed from selected beneficial microorganisms into the liquid larval diet was completed.  Establishment of larval rearing of B. dorsalis on gel based larval diet incorporated with larval gut bacteria spp. was completed.

Gaps  Although the gut bacterial community were identified for laboratory reared flies, the bacterial community of wild flies need to be addressed.  The use of liquid larval diet for large scale mass rearing of B. dorsalis was not found feasible.

Recommendations  Some gut bacterial isolates under Enterobacteriaceae family viz., Enterobacter sp. Proteus sp. Pantoea spp., Serratia sp. can be use as probiotics into larval diet to improve the quality parameters of B. dorsalis.  Gel based larval diet incorporated with Enterobacter and Proteus spp. can be used for future mass scale rearing of B. dorsalis.

Participants: Changying Niu (China)

5 years plan  Identification of microbial communities in egg, larvae and pupae of wild fruit flies by culture-dependent and molecular approaches (16S rRNA pyrosequencing)  Identify suitable beneficial microorganisms for probiotic applications at larval stage.

Achievements at the end of CRP:  Bacterial strains have been isolated from wild fruit flies using culture-dependent approaches, and tested for beneficial traits.  The bacterial diversity of wild populations was characterised using 454 pyrosequencing technology to characterize the diversity and abundance of the bacteria across different life stages. Pyrosequencing analysis reveals a shift in symbiotic bacteria populations in different life stages of B. dorsalis. The most abundant families were Enterococcaceae and Comamondaceae.  GFP tagged Enterobacter cloacae and Enterococcus faecalis was found to form bacterial films 48 hours after ingestion. Additionally, E. faecalis was found present throughout the life cycle.

 Diets enriched with Enterococcus phoeniculicola ended up with lower larval development duration, higher pupal weight, and increased survival in a long term cultured genetic sexing strain of B. dorsalis.

Gaps  Bacterial diversity from wild populations (different area, different host plants).  Localization of spatial-temporal patterns of bacteria in B. dorsalis.

Recommendations  Use in situ localization for bacterial localization purposes.

Participants: George Tsiamis (Greece) Collaborators: Kostas Bourtzis (IAEA)

5 years plan  Characterization of the gut associated bacterial communities by 16S rRNA amplicon libraries

Achievements at the end of CRP:  Bacterial diversity from a lab colony at three developmental stages was estimated using a MiSeq amplicon sequencing approach. Propiniobacterium sp. is the dominant taxa during the larval and 1-day old adults. Lactococcus sp. dominate’s the 5- and 15- day adults.  No difference in the bacterial profile has been detected between males and females.

Gaps  Characterization of gut microflora from natural populations.

Recommendations  Characteriazation at species or strain level for the dominant OTUs will be useful using an MLST and/or genomics approach.

Species: Bactrocera minax Participants: Changying Niu (China)

5 years plan  Identification of microbial communities in egg, larvae, pupae and adults from the wild populations by culture-dependent and molecular approaches (16S rRNA pyrosequencing)  Identify suitable beneficial microorganisms for probiotic applications at larval stage.  Monitor the population dynamics of some important bacteria species identified by culture-dependent approaches Achievements at the end of CRP:  Pyrosequencing results revealed a total of 162 bacterial operational taxonomic units (OTUs) associated with all the life stages of B. minax. About 80% of the reads in all life stages belonged to the Proteobacteria，which represented the most dominant phylum.

 The sugar-acid composition of the different varieties of citrus varied significantly over time as the citrus fruit matures.  The bacteria from the gut of adults, the ovipositor of females, the laying-egg mark of infested fruit, and young larvae were isolated and identified. The results suggested a possible vertical transmission pattern.  Supplementing Klebsiella oxytoca and Citrobacter freundii respectively and cocktail bacteria enriched diets improved the survival rate of young hatched larvae significantly.

Gaps  Vertical and horizontal transimission pattern of specific bacteria in B. minax.  Test cocktail bacteria enriched diet to improve the survival of the newly hatched larvae.

Recommendations  Use FISH technique to localize the bacteria in B. minax.

Species: Bactrocera tryoni Participants: Olivia Reynolds (Australia) Collaborators: Ania Deutscher; Toni Chapman

5 years plan  Identification of microbial communities in B. tryoni larvae of wild and lab reared fruit flies (different strains and colonies) by molecular approaches (16S rDNA amplicon sequencing).  Identify suitable beneficial microorganisms for probiotic applications at the larval stage for B. tryoni. Achievements at the end of the CRP  Characterisation of the bacteria in the larval gut of B. tryoni; Asaia is likely to be an important gut bacterium in B. tryoni larvae.  Identification of the presence of yeast, and yeast-like fungi in the gut of wild larval B. tryoni; first report of yeasts in the gut of B. tryoni.  Identification of several potential larval B. tryoni probiotic candidates.  Impacts of individual and a blend of bacteria fed to larval B. tryoni on various larval and adult traits.

Gaps  Understanding the functional role of bacteria.

Recommendations  Need to consider fungi and bacteria in future microbial symbiont studies.  Consider engaging with a commercial company to produce the larval/adult probiotic.  Analyse individual fly samples, rather than pool samples.

Species: Diorixa pornia Participants: Peter Crisp, Kala Bhandari (Australia)

5 years plan  Identification of microbial communities in egg, larvae and pupae of wild and lab reared fruit flies (different strains and colonies) using molecular approaches (16S rDNA amplicon sequencing).  Identify suitable beneficial microorganisms for probiotic applications at larval stage.  Develop protocols for the incorporation of beneficial bacterial into the larval diet. Achievements at the end of the CRP  Culturable bacteria from the digestive tract and reproductive organs (female) of Dirioxa pornia were isolated and identified using 16S rRNA sequencing.  Adult D. pornia, Ceratitis capitata and Bactrocera tryoni were exposed to agar plates inoculated with bacterial isolates to assess attraction of the flies to the bacteria (Cultures were 3, 50+ and 100+ generations domesticated respectively).  Three of the bacteria (Enterobacter aerogenes, Enterobacter sp. and Citrobacter fruendi) were highly attractive to all three species when supplied as a dietary supplement on agar.  When supplied as a dietary supplement to adults at eclosion both Enterobacter sp. were found to significantly increase the fecundity of female D. pornia and B. tryoni and resulted in increased next generation adult eclosions.  Another of the bacteria isolates (Bacillus amyloliquefaciens) fed to adults at eclosion significantly delayed egg laying in D. pornia and reduced total fecundity by greater than 60%.  The bacterial community in the entire intestinal tract of 12 individual D. pornia has been sequenced using amplicon sequencing approach. All 12 flies were collected from the same tree on the same day.  Addition to the larval diet was not successful and probiotics were used to supplement adult diet at eclosion, bacteria were supplied through inoculation of YEA plates or through cotton wicks placed in nutrient solution.  3D imaging has been completed and results provided in the form of a paper to the special edition.

Gaps  Dissection and identification of bacterial communities in various regions of the gut, egg, oesophageal diverticulum, mid/gut and crop using Illumina has not been completed and is ongoing, results will be published in a refereed journal on completion.

1.1 Summary of the achivements at the end of the CRP In order to reduce mass-rearing costs characterization of beneficial microorganisms for probiotic applications has been accomplished for the majority (shall we mention the species?) of the insects examined. Characterization of the beneficial microorganisms has been completed using culture-dependent and independent approaches. A comprehensive list with 22

putative beneficial microorganisms has been compiled. Protocols for the addition of beneficial bacterial strains to the larval diet have been developed for: C. capitata / Raoultella ornithinolitica, Klebsiella sp., Enterobacter sp. A. ludens / Raoultella ornithinolitica B. dorsalis / Enterobacter cloacae and Enterococcus faecalis, Proteus mirabilis B. minax / K. oxytoca, C. freundii B. tryoni / Asaia sp., Leuconostoc sp., Lactobacillus sp., Enterobacter sp. D. pornia / Enterobacter sp. & B. amyloliquefaciens

1.2. Preventing the growth of deleterious microorganisms

Species: Ceratitis capitata Participants: Hernán Donoso, Carolina Yánez (Chile)

5 years plan  Identification of deleterious microorganisms in laboratory eggs, larvae and larval diet by molecular and classical microbiological approaches.  Development of diagnostic tests for the early detection of deleterious microorganisms.

Achievements at the end of the CRP  Morganella sp. and Stenotrophomonas sp. were detected in eggs and larvae.  The presence of Bacillus sp. was detected in the larval diet.  Screening for assessing microbiological contamination in order to detect potential pathogens in the mass-rearing facility indicated the presence of Morganella sp. (trays) and Stenotrophomonas sp. (trays and bubble stones).  A microbiologist has just been hired at the mass-rearing facility, thus microbiological aspects will be improved in the future. Gaps  Development of diagnostic tests for the early detection of deleterious microorganisms.

Recommendations  Develop diagnostic test for detection of deleterious microorganisms that can be easily apply in mass-rearing facilities (easy and fast). Methods should include culture-dependent and -independent methods.  Unify diagnostic tests in all mass-reared facilities.

Participants: Dina Melgar, Pedro Rendón, Felipe Jerónimo (Guatemala)

5 years plan  Identification of deleterious microorganisms in laboratory eggs, larvae and larval diet by molecular and classical microbiological approaches. Achievements at the end of the CRP  Morganella sp., Serratia sp., Providencia sp., and Pseudomonas aeruginosa have been identified as potential pathogenic microorganisms.  Protocols to evaluate the pathogenic effect in mass rearing and release facility have been developed.

Recomendations  Data base collection of microorganism with the beneficial and pathogenic effects in different fruitfly species.

Participants: Jaime Garcia de Oteyza. Teresa Navarro (Spain) Collaborators: George Tsiamis (Greece)

Achievements at the end of the CRP  Morganella sp., Providencia sp. and Serratia sp. have been identified as potential deleterious microorganisms.  Evaluation of specific primers for the detection of Morganella sp. is in progress.  Strain collection (medfly isolates) is available. Gaps  Evolution of the presence of the bacteria in the larvae and larval diet.  Access pathogenicity of different strains isolated.  Develop pathogenicity theresholds for the mass rearing facilities.

Species: Anastrepha ludens Participants: Dina Melgar (Guatemala)

5 years plan  Identification of deleterious microorganisms in laboratory eggs, larvae and larval diet by molecular and classical microbiological approaches.

Achievements at the end of the CRP  Morganella sp., Serratia sp., Providencia sp., and Pseudomonas aeruginosa have been identified as potential pathogenic microorganisms.  Protocols to evaluate the pathogenic effect in mass rearing and release facility have been developed.

Gaps  Identification of microbial communities in egg, larvae and pupae of wild and mass reared medfly (different strains and colonies), wild infested fruits and mass rearing larval diet by molecular (DGGE, 16S rRNA sequencing) and classical approaches has been not completed.

Recomendations  Database collection of microorganism with the beneficial and pathogenic effects in different Fruitfly species.  Development of aseptic protocols for disinfection of the egg incubation system, larval and pupae trays, pupae maturation and in the release facility.

Participants: Erin Schuenzel, Bacilio Salas, Hugh Conway, Don Vacek (USA)

5 years plan  Identification of deleterious microorganisms in mass-reared eggs, larvae and larval diet by molecular and classical microbiological approaches.  Development diagnostic tests for the early detection of deleterious microorganisms.

Achievements at the end of the CRP  400 isolates were tested for their effect on larval survivorship. Four bacterial species and one fungal species were found to be pathogenic.  For two of the pathogenic bacterial species (Morganella morganii and Morganella pyschrotolerans) a real-time PCR diagnostic based on the hdc gene has been developed but not implemented.  Twenty-two isolates of one the pathogenic bacterial species (Morganella morganii) have been further genotyped with five additional genes. The genotypes were linked to pathogenic effects.  Genomes for three strains of the bacterial species with known pathogenic effects (Morganella morganii, Providencia alcalifaciens, Providencia rettgeri) and two strain of the bacterial species with known non-pathogenic effects (Morganella morganii, Providencia sp.) were sequenced but still need to be aligned and annotated. Gaps  Further genotyping of Providencia alcalifaciens and Providencia rettgeri need to be done to better understand the link between pathogenicity and genotype.  Further genome sequencing needs to be completed on pathogenic and non-pathogenic strains to identify possible pathogenicity island/genes. Recommendations  Further genotyping and genome sequencing of pathogens needs to be completed. Comparison between strains of the same species found in/on other tephritids.

Species: Anastrepha obliqua Participants: Emilio Hernández, Pablo Liedo (México)

5 years plan  Identification of deleterious microorganisms in laboratory eggs, larvae and larval diet by molecular and classical microbiological approaches.

 Development diagnostic tests for the early detection of deleterious microorganisms.

Achivements at the end of the CRP  Molecular identification was completed with primers: Fg16NF/R F. graminearum, FP82F/R for F. oxysporum, FACF/R for F. acuminatum and J1Af for F. avenaceum.  The molecular characterization of Morganella spp. and Serratia spp., was completed using primer ERIC1 (5'-TGAATCCCCAGGAGCTTACAT-3') y ERIC2 (5'- AAGTAAGTGACTGGGGTGAGCG-3') and pyrosequencing.

 Lowest larval yields were obtained with corn cob with the presence of Fusarium graminearum, Morganella morganii and Serratia marcescens.

Gaps  Lethal concentrations should be determined for Fusarium graminearum, Morganella morganii and Serratia marcescens in the larval diet.

Recommendations  It is necessary to determine the lethal concentrations for Anastrepha flies since only in this study was performed to identify the microorganisms in the corn cob, but not to define the lethal concentration of the microorganism and/or of the toxins.

Species: Zeugodacus cucurbitae Participants: Mahfuza Khan (Bangladesh) Collaborators: George Tsiamis (Greece) and Kostas Bourtzis (IAEA)

5 years plan  Identification of deleterious microorganisms in laboratory (host/artificial) reared larvae of B. cucurbitae by culture-dependent and molecular approaches.  Assess the quality parameters of B. cucurbitae reared from bacteria incorporated larval diets.

Achivements at the end of the CRP  Identification of deleterious microorganisms in laboratory host reared larvae of Z. cucurbitae by biochemical approaches (API kits) has been completed.  Rearing of Z. cucurbitae on liquid larval diet has been established.

Gap  No further action was taken to remove the deleterious microorganisms.

Species: Bactrocera dorsalis complex Participants: Mahfuza Khan (Bangladesh) Collaborators: Kostas Bourtzis (IAEA) and George Tsiamis (Greece)

5 years plan  Identification of deleterious microorganisms in laboratory (host/artificial) reared larvae of B. dorsalis by culture-dependent and molecular approaches.  Assess the quality parameters of B. dorsalis reared from bacteria incorporated larval diets.

Achievements at the end of the CRP  Identification of deleterious microorganisms in artificial larval diet by biochemical approaches (API kits).  Assessement on the quality of B. dorsalis reared from four bacteria strains incorporated into the larval diet has been completed.

Gaps

 No further action was taken to remove the deleterious microorganisms.

Participants: George Tsiamis (Greece) Collaborators: Kostas Bourtzis (IAEA)

5 years plan  Identification of (deleterious) microorganisms in laboratory larvae by 16S rRNA amplicon sequencing libraries using NGS technologies.

Achievements at the end of the CRP  Providencia sp. as a putative deleterious organismsm have been identified.

Gaps  Identification at species/strain level is required to identify pathogenic strains.

Recommendations  Develop MLST system and/or PCR detection systems.

Species: Diorixa pornia Participants: Peter Crisp (Australia)

5 years plan  Identification of deleterious microorganisms in laboratory eggs, larvae and pupae using molecular approaches (16S rRNA pyrosequencing).  Development of diagnostic tests for the early detection of deleterious microorganisms.

Achievements at the end of the CRP  Potentially pathogenic bacteria isolated from the gut of D. pornia have been identified and assessed by addition to diet of adults and larval diet. Gaps  ELISA tests are still being developed as are DNA chip technologies for use in mass production facilities.

1.2 Summary of the achivements at the end of the CRP

The presence of deleterious microorganisms is a common problem for all mass rearing facilities. Identification of potential deleterious microorganisms is an essential step for preventing growth and increasing the efficiency in mass rearing facilities. Potential deleterious organisms like, Morganella sp., Stenotrophomonas sp., Serratia sp., Providencia sp., Pseudomonas aeruginosa, Fusarium graminearum, F. oxysporum, F. acuminatum and F. avenaceum have been identified in the insect species examined. For the bacterial isolates identification at species/ strain level is required.

1.3. Improving mass rearing efficiency and quality of the insects produced

Species: Ceratitis capitata Participants: Hernán Donoso, Carolina Yáñez (Chile)

5 years plan  Identification of beneficial microorganisms in laboratory larvae by molecular and classical microbiological approaches.  Development of diagnostic tests for the detection of beneficial microorganisms.  Development of protocols for probiotic introduction in larval diet.

Achievements at the end of the CRP  The bacterial communities in release and injection colonies (larvae, pupae, adults) have been characterized using DGGE.  Bacteria have been isolated from release and injection colonies (larvae, pupae, adults).

Gaps  Development of protocols for probiotic introduction in larval diet.  Quality assessment of the bacterial fed larvae  Development of diagnostic tests for the detection of beneficial microorganisms.

Recommendations  Develop diagnostic test for detection of deleterious microorganisms that can be easily applied in mass-rearing facilities (easy and fast). Methods should include culture- dependent and -independent methods.  Unify diagnostic tests in all mass-reared facilities.

Participants: Dina Melgar, Pedro Rendón, Felipe Jerónimo,

5 years plan  Identification of beneficial microorganisms in laboratory eggs, larvae and larval diet by molecular and classical microbiological approaches.  Monitoring beneficial microorganisms in egg suspension.  Development of protocols for the incorporation of beneficial microorganisms in the egg suspension.

Achievements at the end of the CRP  Development of protocols for the incorporation of beneficial microorganisms.  Periodic monitiring for the presence of beneficial microorganismsm in egg suspension.  Middle-scale trials have been completed in mass rearing and release facilities.  Development of protocols for the incorporation of beneficial microorganisms.  Beneficial microorganisms have been identified.  Protocols to avoid the pathogenic bacteria have been developed.  Middle-scale trials have been completed.

Gaps  Identification of beneficial microorganisms in laboratory eggs, larvae and larval diet by molecular and classical microbiological approaches.

Recommendations  Development of general protocols for beneficial bacteria introduction/applying in mass rearing and reléase facilities.  Introduction of wild beneficial bacteria in mass rearing facilities and release. Decypher genomic sequence.  Tipification of microorganisms when the mass rearing insect strain is refreshed with wid material.

Participants: Jaime Garcia de Oteyza, Teresa Navarro (Spain) Collaborators: George Tsiamis (Greece)

5 years plan  Identification of beneficial microorganisms in laboratory eggs, larvae and larval diet by molecular and classical microbiological approaches.  Development of diagnostic tests for the detection of beneficial microorganisms.  Protocol development for introducing beneficial microorganisms in a mass-rearing facility.

Achivements at the end of the CRP  Potential beneficial microorganisms have been identified using molecular and culture- dependent approaches.  Small scale-trials have been completed.  Protocol for probiotic supply has been evaluated. Gaps  Determine the most effective probiotic supply time.

Species: Anastrepha ludens Participants: Dina Melgar (Guatemala)

Achievements  Development of protocols for the incorporation of beneficial microorganisms has been developed.  Periodic monitiring for the presence of beneficial microorganismsm in egg suspension.  Middle-scale trials have been completed in mass rearing.  Protocols to avoid the pathogenic bacteria have been developed.

Gaps

 Identification of beneficial microorganisms in laboratory eggs, larvae and larval diet by molecular and classical microbiological approaches.

Recommendations

 Development of general protocols for beneficial bacteria introduction/applying in mass rearing and reléase facilities.  Introduction of wild beneficial bacteria in mass rearing facilities and release. Decypher genomic sequence of beneficial bacteria.  Tipification of microorganisms when the mass rearing insect strain is refreshed with wid material.

Participants: Emilio Hernández, Pablo Liedo (México)

5 years plan  Identification of beneficial microorganisms in laboratory eggs, larvae and larval diet by molecular and classical microbiological approaches.  Development of protocols for the incorporation of beneficial microorganisms in the egg suspension

Achievements at the end of the CRP  Identification of microbial communities from eggs, larvae and larval diet by classical methods are finished but the molecular identification (pyrosequencing) is finalized but not published.

Gaps  The results of the identification should be analysed and published.  The evaluation of the effect of the microbiota associated to the eggs, larvae and larval diet should be determined on the life history traits, quality and mass rearing parameters.  Anastrepha ludens Tap-7, a sexing strain, should be studied.

Recommendations  Finalize the identification and publish the results of the microbiota associated to the eggs, larvae and larval diets.  Continue with evaluation of the effect of the microbiota associated to the eggs, larvae and larval diet on the life history traits, quality and mass rearing parameters.  Isolated and identify the microbiota associated to the eggs incubation, larvae and larval diet of Anastrepha ludens Tap-7, a sexing strain.

Participants: Erin Schuenzel, Bacilio Salas, Hugh Conway, Don Vacek (USA)

5 years plan  Identification of beneficial microorganisms in laboratory eggs, larvae and larval diet by molecular and classical microbiological approaches.  Development diagnostic tests for the detection of beneficial microorganisms.

Achievements at the end of the CRP

 No beneficial bacteria were identified with the small cup tests for the over 400 isolates tested. Recommendations  Small-cup tests may not be the correct way to evaluate beneficial bacteria. A new methodology needs to be developed.

Species: Anastrepha obliqua Participants: Emilio Hernández, Pablo Liedo (México)

5 years plan  Identification of beneficial microorganisms in laboratory eggs, larvae and larval diet by molecular and classical microbiological approaches.  Development of protocols for the incorporation of beneficial microorganisms.

Achivements at the end of the CRP  An enriched larval diet was formulated for A. obliqua adding Lactobacillus rhamnosus, which survived very well on artificial larval diet during 8 days maintaining a concentration of 3.4 x 108 cfu/g during first 5 days, which means the L. rhamnosus was able to survive and reproduce. After 5 days, the concentration was decreasing rapidly to reach 0.53 x 107 cfu/g. The enriched larval diet results in increased survival of the larva from 80 to 90%, increased from 3 to 4 larval recovery per gram of diet, increased in 1-mg the pupal weight, increased the fliers from 90 to 94%.

Gaps  The enriched larval diet with L. rhamnosus should be used in mass rearing conditions.  Molecular indetification of the gut bateria are not finished, it is outgoing.

Recommendations  Validate at massive level diets enriched with Lactobacillus rhamnosus for Anastrepha obliqua and A. ludens.  Finalize the molecular indetification of the gut bacteria.

Species: Anastrepha striata

5 years plan  Identification of beneficial microorganisms in laboratory eggs, larvae and larval diet by molecular and classical microbiological approaches.  Development of protocols for the incorporation of beneficial microorganisms.

Achievements at the end of the CRP  An enriched larval diet was formulated for A. striata. The bacteria that were isolated from the digestive tract of wild flies of A. striata corresponded to Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterobacter cloacae and Enterococcus faecalis. The effect of the preservatives, methyl paraben and sodium benzoate at different pH were performed on each species of bacteria to determine their effect on 31

viability. The bacteria were inoculated in a started-diet using the 5x105 and 5x107 CFU/g. The results indicated that the addition of Enterobacter cloacae allows 33% larva-pupa survival, whereas Klebsiella pneumoniae inoculation without metabolites allows the increase the pupa weight from 13.56 to 19.27 mg, survival larva-pupa from 20.33 to 33%.

Gaps  The larval enriched diet with bacteria sould be used to maintain the laboratory colony.

Recommendations  Use the enriched larval diet for colonize and maintain laboratory colony.

Species: Anastrepha fraterculus Participants: Fernando Consoli (Brazil)

5 years plan  Identification of beneficial microorganisms in laboratory larvae and adult reared in natural fruits by molecular (16S rRNA gene sequencing) and classical microbiological approaches.

Achievements at the end of the CRP  Culturable and non-culturable larval and adult gut microbiota of a laboratory population reared on papaya, and natural populations from different host fruits have been identified.

Species: Bactrocera cucurbitae Participants: Mahfuza Khan (Bangladesh)

5 years plan  Identification of beneficial microorganisms in laboratory (host/artificial) reared larvae of B. cucurbitae by molecular/biochemical and culture-dependent approaches.  Assess the quality parameters of B. cucurbitae reared from bacteria incorporated larval diets. Achievements at the end of the CRP  Identification of beneficial microorganisms in artificial larval diet by biochemical approaches (API kits).  Quality parameters were assessed using different beneficial bacterial species.

Species: Bactrocera dorsalis complex Participants: Mahfuza Khan (Bangladesh) Collaborators: Kostas Bourtzis (IAEA) and George Tsiamis (Greece)

5 years plan  Identification of deleterious microorganisms in laboratory (host/artificial) reared larvae of B. dorsalis by culture-dependent and molecular/biochemical approaches.  Assess the quality parameters of B. dorsalis reared from bacteria incorporated larval liquid and gel diets.

Achievements at the end of CRP  Identification of beneficial microorganisms in artificial larval diet by biochemical approaches (API kits).  Quality parameters of B. dorsalis were assessed using different beneficial bacterial species.

Participants: George Tsiamis (Greece) Collaborator: Kostas Bourtzis

5 years plan  Identification of (beneficial) microorganisms in laboratory larvae by 16S rRNA amplicon sequencing libraries using NGS technologies.

Achievements at the end of the CRP  Potential beneficial microorganisms have been identified (Lactobacillus sp.)

Gaps  Bacterial profiles from natural populations are not very well characterised.

Recommendations  A strain identification system should be developed in order to use host specific bacterial species.

Species: Diorixa pornia Participants: Peter Crisp (Australia)

5 years plan:  Identification of beneficial microorganisms in laboratory eggs, larvae and larval diet by molecular approaches.  Development of diagnostic tests for the detection of beneficial microorganisms.

Achievements at the end of the CRP  DNA has been extracted from adults, libraries for 454 amplicon pyrosequencing have been prepared and candidate beneficial bacterial strains have been identified. (List of bacterial OTUs).  Preliminary cage feeding trials have been conducted and three beneficial species selected for further development and mass production protocols.  Culturable bacteria from the digestive tract and reproductive organs (female) of Dirioxa pornia were isolated and identified using 16S rRNA with PCR and sequencing by Australian centre for Functional Genomics. o Adult D. pornia, Ceratitis capitata and Bactrocera tryoni were exposed to agar plates inoculated with bacterial isolates to assess attraction of the flies to the bacteria. (Cultures were 3, 50+ and 100+ generations domesticated respectively). o Three of the bacteria (Enterobacter aerogenes, Enterobacter sp. and Citrobacter fruendi) were highly attractive to all three species when supplied as a dietary supplement on agar.

o When supplied as a dietary supplement at Eclosion both Enterobacter sp. were found to significantly increase the fecundity of female D. pornia and B. tryoni and resulted in increased next generation adult eclosions. o Another of the bacteria isolates (Bacillus amyloliquefaciens) significantly delayed egg laying in D. pornia and reduced total fecundity by greater than 60%. o When provided to irradiated C. capitata in the 48 hours after eclosion the treated flies were observed to be more active and dispersed more quickly than flies supplied with agar and sugar.  The bacterial community in the entire intestinal tract of 12 individual D. pornia has been sequenced using NGS. All 12 flies were collected from the same tree on the same day. o Of these 8 (5 female and 3 male) were of a suitable quality for analysis. o There communities appeared to differ among flies but is yet to be statistically analysed.  Potentially identified bacteria isolated from the gut of D. pornia have been identified and assessed by addition to diet of adults and larval diet. Gaps  ELISA tests are still being developed as are DNA chip technologies for use in mass production facilities.

1.3 Summary of the achivements at the end of the CRP Implementation of the protocols developed at 1.1 has been done for C. capitata, A. ludens, A. obliqua, A. striata, B. tryoni, and B. dorsalis by performing small and middle scale trials. For C. capitata inclusion of Enterobacter sp. in the larval diet has successfully replaced the need of yeast as protein source. A cost-benefit analysis for the technology transfer to the mass- rearing facilities remains to be completed. Several benefits of the addition of probiotics to the larval diet have been recorded including reduced larval development time, increased larval recovery (number of larvae/ g of diet), pupal weight, increased adult longevity, increased flight ability, male sex ratio and increased locomotor activity.

2 Probiotics

Background Symbiotic bacteria are ubiquitous in tephritid fruit flies. In the various genera of these flies similar microbial communities are generally found. A number of studies suggest that microorganisms contribute to the fitness of their insect host including the reproductive success of males. Many studies have shown that mass-reared sterile males are clearly disadvantaged compared to wild males in both survival and sexual competitiveness. Apparently, the reasons for this low competitiveness stems from their colonization, mass-rearing conditions and the irradiation process. In order to compensate for their low competitiveness, sterile flies are released in large numbers relative to the number of wild flies present in the orchard (in ratios up to 100:1). This over flooding has economical and operational consequences. Therefore, there is a real need to improve the competitive ability of the released sterile males in SIT operations. Several approaches have been developed for this purpose. Chief amongst these have been manipulations of the olfactory environment of the sterile males (Papadopoulos et al., 2001; Shelly et al., 2005). Indeed, the exposure of adult flies to the aroma of ginger root oil improves sexual competitiveness and is being implemented in some SIT operations (Shelly et al., 2007). Other approaches, have been promising in laboratory or semi field conditions, but have yet to be applied on operational scales. These include manipulation of the pre-release diet (Yuval and Hendrichs 2000; Yuval et al., 2002) and juvenile hormone treatments (Teal et al., 2000; 2007). The complement of bacteria present in released males following mass-rearing and irradiation may differ from their wild counterparts enough to impede their performance. There is evidence that inoculating symbiotic bacteria, prior to release, can significantly improve male sexual performance. Thus, restoring key bacteria to mass-reared sterile flies prior to their release is a valid approach to improve the efficacy of the SIT. It is worthwhile to validate this approach at an operational level and to extend it to other insects targeted by the SIT.

Subtheme A: Structure of microbiota

Current knowledge:

A1. General: The association between fruit flies and bacteria was first recognized in the beginning of the twentieth century, when, based on microscopic observations, Petri described symbiotic relations between the olive fly Bactrocera oleae and a microorganism. He suggested that this symbiont might be Pseudomonas savastanoi, a bacterial pathogen causing the olive knot disease (Petri 1909). Bacterial isolation from fruit flies and their oviposition sites were sporadically reported since 1934. However, beginning in the mid-1980s many studies were conducted on fruit fly – bacterial interactions (see reviews by Drew & Lloyd 1987, 1991; Lauzon 2003). These studies

were based on traditional microbial methods such as viable plate counts and phenetic taxonomy and focused mainly on the bacteria inhabiting the digestive system of adult fruit flies (Table 2). Two species received quite a lot of attention – the Queensland fruit fly, Bactrocera tryoni, and the apple maggot fly Rhagoletis pomonella. The bacteria found to be associated with B. tryoni were members of the family Enterobacteriaceae, mainly species of Klebsiella and Enterobacter, with Klebsiella oxytoca and Enterobacter cloacae as the most common species. These bacteria were also found in the different instars of the flies and in infested fruit (Fitt & O'Brien 1985; Drew & Lloyd 1987; Table 2). Species of Klebsiella and Enterobacter are also commonly associated with R. pomonella. Lauzon described different associations, mainly with Enterobacter agglomerans and Klebsiella pneumoniae (Lauzon 2003). Howard et al. (1985) found that Klebsiella oxytoca is the most common species associated with this fly throughout its life and suggested that this bacterium might be the equivalent symbiont in R. pomonella, to Pseudomonas savastanoi in the olive fly (Howard et al. 1985) (Table 2). However, the use of the 16S rRNA approach has redefined the microbial community associated with the olive fly. Capuzzo et al. (2005) showed that the olive fly's symbiont is not Pseudomonas savastanoi (as suggested by Petri (1909)), and proposed a novel bacterial species: Candidatus Erwinia dacicola.

A2. Species level background: The following table summarizes studies on the microbial communities of tephritid fruitflies. Classical methods of identification include biochemical, culture and/or microscopic techniques. Molecular techniques for identification include 16S rRNA community sequencing, 16S rRNA Sanger sequencing, DGGE and/or RFLP analysis. Community-based analyses have increased the known diversity of the microbiota of the fruit flies.

Table 2. Summary of the studies on the microbial communities of tephritid fruit flies. (Not exhaustive)

Tephritid Source of Bacterial species Microbial Reference species: sample: found (most common Method species in bold): Anastrepha Adult gut Enterobacter cloacae, Classical Kuzina et al. ludens Providencia spp 2001 Citrobacter koseri Enterobacter sakazakii Klebsiella pneumoniae Pseudomonas aeruginosa A. ludens Adult crop and Citrobacter freundii Classical Martinez et al. gut Klebsiella oxytoca 1994 A. ludens Adult crop Enterobacter spp. Classical Martinez et al. Pseudomonas aeruginosa 1994 Pseudomonas spp. A. ludens Adult gut Klebsiella pneumoniae Classical Martinez et al. 1994 A. ludens infested fruit Citrobacter freundii Classical Martinez et al. Klebsiella oxytoca 1994 Bactrocera Adult gut, Citrobacter freundii Classical Fitt & Obrien cacuminata Pupae, Eggs, Klebsiella pneumoniae 1985 infested fruit Pseudomonas spp B. cacuminata Host Plant Pantoea spp Classical Raghu et al. 2002

leaves and Pantoea agglomerans fruit surface Citrobacter Adults Enterobacter Thaochan et al. Klebsiella 2010 Providencia Serratia Molecular Morrow et al., Enterococcus 2015 Orbus Vagococcus

B. cucurbitae Adult mid-gut Klebsiella oxytoca Molecular Hadapad et al., Citrobacter freundii (16S rRNA 2015 Providencia rettgeri gene) B. dorsalis Adult gut KlebsiellaCitrobacter Molecular Wang et al. 2011 Enterobacter Pectobacterium Serratia Actinobacteria Firmicutes B. jarvisi Adult gut, Enterobacter Classical Fitt & Obrien Pupae, Eggs, agglomerans 1985 infested fruit Enterobacter cloacae Enterobacter spp Klebsiella pneumoniae Providencia spp B. jarvisi Adult gut, Pseudomonas spp. Classical Fitt & Obrien Pupae, Eggs, Enterococcus 1985 infested fruit Lactococcus Morrow et al., Serratia Molecular 2015 Vagococcus B. minax Adult gut, Molecular Wang et al., 2014 reproductive organs B. neohumeralis Adult gut, Enterobacter cloacae Classical Fitt & Obrien Pupae, Eggs, Enterobacter spp 1985 infested fruit Pseudomonas spp Morrow et al., Acetobacter Molecular 2015 Dysgonomonas Enterococcus Escherichia/Shigella Kluyvera Lactobacillus Lactococcus Providencia Serratia Vagococcus B. oleae Larvae Ca. Erwinia dacicola Molecular Ben-Yosef et al., (16S rRNA 2015 gene) B. oleae Adult Ca. Erwinia dacicola Molecular Capuzzo et al. oesophageal (16S rRNA 2005 bulb, gut, gene) Sacchetti et al., ovipositor 2008

B. oleae Adult Pseudomonas savastanoi Classical Petri 1909 oesophageal bulb B. oleae Adults Acetobacter tropicalis Molecular Kounatidis et al., 2009 B. oleae Adults Ca. Erwinia dacicola Molecular Estes et al., 2012 Ben-Yosef et al., 2014 B. tau Adults Enterobacteriaceae Classical & Prabhakar et al. Staphylococcus Molecular 2009 B. tryoni Adult crop, Enterobacter cloacae Classical Drew & Lloyd gut, Klebsiella oxytoca 1987 mouthparts; Klebsiella ozaenae host plant Pantoea agglomerans Providencia spp Acetobacter Thaochan et al., Adults Asaia 2010 Dysgonomonas Molecular Morrow et al., Enterococcus 2015 Escherichia/Shigella Kluyvera Lactococcus Orbus Serratia Vagococcus B. tryoni Adult gut, Enterobacter Classical Fitt & O'Brien Pupae, Eggs, agglomerans 1985 infested fruit Enterobacter cloacae Klebsiella pneumoniae Providencia spp Pseudomonas spp B. tryoni Adult crop, gut Klebsiella oxytoca Classical Murphy et al. Enterobacter cloacae 1988; 1994 B. tryoni Adults Serratia Molecular Thaochan et al., 2010 Ceratitis capitata Adult gut Enterobacter spp. Classical Lauzon 2003 Klebsiella spp. C. capitata Adult Enterobacter Classical Marchini et al. oesophageal agglomerans 2002 bulb Klebsiella oxytoca Enterobacter cloacae Pseudomonas putida Pseudomonas spp C. capitata Adult gut, Enterobacter Molecular Behar et al., larvae, pupae, Klebsiella 2005, 2008a,b eggs, host Pseudomonas plant Pectobacter Morrow et al., Pantoea 2015 C. capitata Adults, larvae Kluyvera Molecular Aharon et al., Leuconostoc 2013 Dirioxa pornia Adult mid-gut Acetobacter Molecular Morrow et al., Asaia 2015 Enterobacter

Gluconacetobacter Kluyvera Lactobacillus Rhagoletis Adult gut, Enterobacter spp Classical Daser & Brandl alternata Larvae Erwinia spp 1992 R. completa Adult Klebsiella oxytoca Classical Howard et al. Oesophageal Klebsiella ozaenae 1985 bulb Klebsiella pneumoniae R. cornivora Adult Klebsiella oxytoca Classical Howard et al. Oesophageal Klebsiella pneumoniae 1985 bulb R. electromorpha Adult Klebsiella oxytoca Classical Howard et al. Oesophageal Enterobacter cloacae 1985 bulb Klebsiella ozaenae Klebsiella pneumoniae R. mendax Adult Klebsiella oxytoca Classical Howard et al. Oesophageal 1985 Enterobacter bulb agglomerans Enterobacter cloacae Klebsiella ozaenae Klebsiella pneumoniae R. pomonella Adult Klebsiella oxytoca Classical Howard et al. Oesophageal Enterobacter 1985 bulb agglomerans Enterobacter cloacae Klebsiella ozaenae Klebsiella pneumoniae Pseudomonas putida Pseudomonas spp R. pomonella Adult Crop, Enterobacter Classical Lauzon 1998; gut, agglomerans 2002 oesophageal bulb R. pomonella Adult gut Klebsiella pneumoniae Classical Lauzon 1998; 2002 R. pomonella Adult Enterobacter cloacae Classical Rossiter et al. Oesophageal 1983 bulb R. pomonella Adult Klebsiella oxytoca Classical Rossiter et al. Oesophageal 1983 bulb, Eggs, Larvae, Pupae, infested fruit R. suavis Adult Klebsiella oxytoca Classical Howard et al. Oesophageal Enterobacter 1985 bulb agglomerans Enterobacter cloacae Klebsiella ozaenae Klebsiella pneumoniae R. tabellaria Adult Klebsiella oxytoca Classical Howard et al. Oesophageal Enterobacter 1985 bulb agglomerans Enterobacter cloacae

Klebsiella ozaenae Klebsiella pneumoniae Tephritis conura Adult gut, Erwinia spp Classical Daser & Brandl Larvae 1992 Tephritis Adult gut, Enterobacter spp Classical Daser & Brandl dilacerate Larvae 1992 Urophora Adult gut, Erwinia spp Classical Daser & Brandl cuspidata Larvae 1992 Urophora Adult gut, Erwinia spp Classical Daser & Brandl solstitialis Larvae 1992

In addition to these published reports, several unpublished results have been reported in this meeting listed in Table 4.

Gaps identified:

A1. General: Many of the previous studies have relied on numerous biochemical and molecular techniques to identify the bacterial communities. Because of the variety of methods and collection strategies of the fruit fly populations, confounding results may be clouding the literature. Standard practices for identification of microbial communities and specific microbes need to be established. Additionally, many of the studies focused on biochemical methods of bacterial identification, which are not as accurate as molecular techniques, specifically 16S rRNA sequencing. The fruit fly populations under study can also result in very different microbial communities. When evaluating the microbial communities, the identity of the flies will create a bias in the community. The following variables have been identified as important considerations in sampling design:  Strain: laboratory versus wild-caught  Age  Sex  Larval Environment – rearing media (lab) and plant host (wild)  Adult Environment – plant host (wild)  Nutritional Status and Diet  Season and Environmental Conditions  Temperature – rearing conditions  Fly tissue sampled  Irradiation  Exposure to Antibiotics and Preservatives

The current literature provides adequate coverage of bacterial communities. To fully appreciate the role bacterial communities play in respect to environmental conditions, functional understanding needs to be reached. Also other domains of microorganisms need to be investigated including Archaea, Eukaryotes such as fungi and protists as well as viruses.

A2. Species level background

Anastrepha: Reproductive endosymbionts for A. striata and A. ludens have been characterized. Community structures of lab-reared and wild A. grandis, A. ludens, A. fraterculus (3 populations – Peru, Tucuman, Brazil) have been studied. Bactrocera: Most species are lacking molecular data except B. dorsalis, B. oleae, B. tau and B. tryoni, B. cucurbitae. For the relatively well-studied olive fly, the differences in community structure of wild and lab-reared flies need to be resolved. Ceratitis: Only the medfly has been studied. Dacus: Molecular data is lacking for the entire genus. Dirioxa: Several individual flies have been molecularly characterized for D. pornia. Molecular data is lacking for the rest of the genus. Rhagoletis: Molecular data is lacking for the entire genus.

Subtheme B: Effects of radiation

Current knowledge

B1 General In addition to the changes induced by colonisation, irradiation (and probably sterilisation by other means) affects the structure of the microbial community (Ben-Ami et. al., 2010; Lauzon and Potter 2012).

B2. Species level background Anastrepha: Irradiation harms the epithelia of the mid-gut and affects the ability of bacteria to colonize this region (Lauzon and Potter 2011). Ceratitis: The structure of the microbial community in irradiated flies differs significantly from that of mass reared or wild flies. Specifically, the abundance of Enterobacteriaceae is reduced and that of pseudomonads increased.

Gaps identified:

B1. General There is a general lack of understanding of this phenomenon. The main questions to be resolved are —why are some bacteria affected and others not, can the damage to the gut be remediated by probiotic bacteria (see subtheme D below).

B2. Species level background The gaps mentioned above are relevant to all mass reared species and SIT programs that rely on the release of irradiated males.

Subtheme C: Function of microbiota and fitness effects

Current knowledge:

C1. General

The widespread and ubiquitous presence of discrete microbial communities, coupled with specific anatomical modifications in their hosts, suggests that these microorganisms contribute to the fitness of their insect host. In general, bacteria are an important food source for tephritids, but how this is mediated is unclear. To date several specific functions of resident bacteria have been identified. These include nitrogen fixation, pectinolysis, and utilization of urea as a nitrogen source. In addition the properties displayed by secondary symbionts of other insects, such as thermotolerance, insecticide resistance, immunity to pathogens and host range expansion may be present in tephritid microbial communities as well.

C2. Species level background Anastrepha: For A. fraterculus there are data suggesting that removal of gut bacterial communities has an impact on male mating performance. Bactrocera: In the olive fly, the presence of symbionts is obligatory to the development of larvae in olives. Furthermore, symbionts enable adults to utilize nitrogen poor diets thus contributing to female fitness. In the melon fly, Klebsiella oxytoca and Citrobacter freundii were successful at attracting both sexes. The addition of bacteria to the adult sugar diet served as nutritional supplement in B. dorsalis. Ceratitis: Bacteria facilitate the utilization of the host plant by the medfly larvae. In the adult stage bacteria contribute to nutrition, longevity and male mating success. Dacus: No information available Dirioxa: In order to rear under laboratory conditions, D. pornia needs bacterial supplements. Rhagoletis: A nutritional function has been assumed.

Gaps identified:

C1. General Although the community structure of many species has been described, relatively little is known of their function and contribution to fly fitness, and this needs to be addressed in each fly species. Novel tools can be brought to bear such as functional genomic assessment, metatranscriptomics and metagenomics to help answer these gaps.

C2. Species level background Anastrepha: The function and fitness contributions of the microbial communities need to be determined. Bactrocera: For the olive fly, determine the physiological basis of bacterial activity and the possibility to emulate or disrupt the activity artificially. The function and fitness contributions of the microbial communities need to be determined for other species of interest. Ceratitis: Unravel how different members of the described community contribute alone and together to larval development and adult nutrition and reproduction. Determine the physiological basis of bacterial activity and the possibility to emulate the activity artificially. Dacus: The function and fitness contributions of the microbial communities need to be determined.

Dirioxa: Determine the mechanism whereby the bacteria contribute to adult reproduction in D. pornia. In addition to female fitness effects, how adults use and acquire the bacteria need to be understood. The establishment and persistence of bacteria within the insect needs to be determined. Rhagoletis: The function and fitness contributions of the microbial communities need to be determined.

Subtheme D: Applications

Current knowledge:

D1. General The widespread presence of bacterial symbionts suggests that their presence can be manipulated to harm the insect host, or improve its condition. The latter option is relevant to the sterile insect technique, the success of which relies upon sterile males mating with wild females, therefore disrupting reproduction. Bacterial inoculations, provided to the larval or adult stage (or both) could improve the efficiency of mass rearing. Importantly, the performance of sterile males may be improved by providing a symbiotic inoculum prior to release in the field. To date only a few studies have been carried out based on this rationale (detailed below). However their results suggest that this is a valid approach.

D2. Species level background Anastrepha: For A. obliqua and A. serpentina, bacterial supplements to adults had a beneficial impact on matings (Gomez et al. unpublished). For A. fraterculus there are data suggesting that removal of gut bacterial communities has an impact on male mating performance, starvation resistance and nutritional status. Bactrocera: In the olive fly, feeding adults with Pseudomonas putida isolated from the oesophageal bulb of wild flies, showed improvement in subsequent egg production (Sacchetti et al., 2014). In B. dorsalis, Khan et al. (unpublished) observed mating enhancement by adding Klebsiella and Proteus incorporated into a full diet and sugar diet of adults. For B. tryoni and B. tau bacteria added to female diet did not affect fecundity (Meats et al., 2009; Khan et al., 2011) but improved larval viability for B. tryoni (Crisp et al., 2013). Ceratitis: In the medfly, Niyazi et al., (2004) provided a mixture of Enterobacter and Klebsiella to irradiated males. Results obtained in the field were inconsistent, as no differences between treated and control males were found for any of the diets. Ben-Ami et al. (2010) provided sterile males with an oral inoculum of K. oxytoca derived from wild medflies. This strain became established in the flies gut, and preliminary results suggested improvement in sexual performance. Indeed, the latency to mating of sterile males was shorter following feeding on this bacterial strain (Ben-Ami et al., 2010). Gavriel et al (2011) found that enriching the sterile male diet with K. oxytoca significantly improved mating competitiveness in the laboratory and in field cages. In addition, bacterially enriched sterile males inhibited female receptivity more efficiently than sugar fed males and survived longer duration of starvation. Hamden et al (2013) enriched larval diets with Klebsiella, Enterobacter and Citrobacter and found increased male mating competitiveness. Similarly, probiotics application of Enterobacter sp. resulted in improvement in larval productivity and reduced rearing duration (Augustinos et al., 2015).

Dirioxa: Bacteria provided to adults enable successful colonization. Specifically, the provision of bacteria increases activity levels and improves hatching rates (Crisp et al., unpublished).

Gaps identified

D1. General This approach, based on community and function/fitness studies, needs to be brought to bear on additional species of economic importance. As these studies evolve, several important standards should be observed:  Formulation – determine that the formulation provided allows survival, and growth in the insect (growth stage of bacteria harvested, osmolarity of solution, pH, etc). Precise identification of the specific strain of probiotic bacteria using complete 16S rRNA sequencing, MLST (if available) and biochemical tests or other recognized identification methods must be performed.  Koch's postulates- important to recover inoculated symbiont in order to be sure that it is providing the benefit. Suggested positive controls- green fluorescent protein (gfp - OK for experiments, not release) antibiotic resistant bacteria or other genetic markers. Proper negative controls (e.g.- check that autoclaved bacteria do not provide the same benefits as live ones).  Delivery of probiotics – different methods of delivery of probiotics and entomopathogens to adults can alter the production of the next generation of adults. Guidelines should be developed to ensure consistency.  Consortium of bacteria – more studies need to move beyond testing a single bacterium and begin to test consortia of bacteria in adult probiotic diets  Metabolic characterization of bacteria – the probiotic effect may be due to the metabolic capability of the bacterium and not the particular species. When possible the biochemical abilities of the bacterium and the physiological effects on the host should be determined.

D2. Species level background Anastrepha: For most species, no studies have been conducted. In A. obliqua and A. serpentina the nature of the supplemental bacteria as a food source or symbiont needs to be determined. Bactrocera: For most species, no studies have been conducted. In olive flies, determine how combinations of bacteria can improve fitness. Explore the possibility of reintroducing Ca. E. dacicola to laboratory lines. In B. dorsalis, the nature of the supplemental bacteria as a food source or symbiont needs to be determined. Ceratitis: In medflies, determine how combinations of bacteria can improve fitness. Optimal formulation of adult diets and preliminary field trials are needed. Dacus: No studies have been conducted. Dirioxa: In D. pornia, the nature of the supplemental bacteria as a food source or symbiont needs to be determined. Rhagoletis: No studies have been conducted.

Individual Plans

2.1. Structure of microbiota

Genus Anastrepha

Anastrepha ludens Participants: Emilio Hernandez, Pablo Liedo, Marysol Aceituno (Mexico)

5 year plan  Identification of microbial communities in wild and laboratory adult (irradiated and non-irradiated) by using molecular and classical microbiological phenotyping approaches.  Identification of the beneficial bacteria and determine the role of the associated bacteria on the rearing (fecundity, fertility, life span) and behaviour parameters.

Gaps  The results of the identification are not analysed and published.

Recommendations  Finalize the identification and publish the results.

Anastrepha obliqua Participants: Emilio Hernández, Pablo Liedo, Marysol Aceituno (México)

5 year plan  Identification of microbial communities in wild and laboratory adult (irradiated and non-irradiated) by using molecular and classical microbiological phenotyping approaches.  Identification of the beneficial and determine the role of the associated bacteria on the rearing (fecundity, fertility, life span) and behaviour parameters.

Achievements  Identification of microbial communities from wild and laboratory larvae (third instar) and adult populations using molecular approaches (pyrosequencing) is ongoing, finalized but not published.  An enriched larval diet was formulated for A. obliqua adding Lactobacillus rhamnosus, which survived very well on artificial larval diet during 8 days maintaining a concentration of 3.4 x 108 cfu/g during first 5 days, which means the

L. rhamnosus was able to survive and reproduce. After 5 days, the concentration was decreasing rapidly to reach 0.53 x 107 cfu/g. The enriched larval diet results in increased survival of the larva from 80 to 80%, increased from 3 to 4 larval recovery per gram of diet, increased in 1-mg the pupal weight, increased the fliers from 90 to 94%.  An enriched Mubarqui® adult diet was formulated adding the isolated bacteria from the midgut of A. obliqua that corresponded to Providencia rettgeri, Serratia marcescens, Citrobacter freundii, Erwinia spp., Staphylococcus aureus, and Enterobacter cloacae. A. obliqua increased the survival of the adults from 8 to 15 day when the fed with Mubarqui® adult diet enriched with P. rettgeri, E. cloacae and Erwinia spp at the concentration of 5x104 UFC/g results in. In a second experiment the Mubarqui® was reformulated and was added Providencia rettgeri results in that the males fed with food enriched with P. rettgeri increased the mating competitiveness and the captured females during the field cages tests. Seven volatile compounds were identified during the calling of A. obliqua males ((Z) -3-nonenol, nonadienol, sesquiterpene, (E, Z) -α-farnesene, β-farnesene (E, E) -α-farnesene and farnesene isomer.

Gaps  The results of the identification shoud be analysed and published.

Recommendations  Determine the conditions to add of Providencia rettgeri to feed the adults during the sexual maturation for release Anastrepha obliqua, to eliminate any risk for the workers according the regulation of a Moscafrut Safe Facility.  Since Klebsiella sp. was isolated from larva and adult evaluate the addition in the adult food and the effect on the quality parameters for release flies. And complete the identification of the species.  Complete the determination of the phylogenetic diversity of bacteria in larva and adults, wild and mass reared.

Anastrepha serpentina Participants: Emilio Hernández, Pablo Liedo, Marysol Aceituno (México)

5 year plan  Identification of microbial communities in wild and laboratory non-irradiated adults by using molecular and classical microbiological phenotyping approaches.  Determine the role of the associated bacteria on the rearing and behaviour parameters.

Achievements  Identification of microbial communities from wild and laboratory larvae (third instar) and adult populations using molecular approaches (pyrosequencing) is ongoing, finalized but not published.  An enriched Mubarqui® adult diet was formulated adding the isolated bacteria from the midgut of A. serpentine, that corresponded to Serratia marcescens, Pantoea aglomerans, Providencia rettgeri, Staphylococcus aureus, and Chryseobacterium indologenes. Which were added to the larval diet at a concentration of 5x104 UFC/g.

Results indicate that when P. aglomerans, P. rettgeri, and C. indologenes were added there was an increase in the survival of the adults from 7 to 13 days.

Gaps  The results of the identification should be analysed and published.

Recommendations  Finalize the identification and publish the results.

Anastrepha striata Participants: Emilio Hernández, Pablo Liedo, Marysol Aceituno (México)

5 year plan  Identification of microbial communities in wild and laboratory non-irradiation adults by using molecular and classical microbiological phenotyping approaches.  Determine the role of the associated bacteria on the rearing and behaviour parameters.

Gaps  The results of the identification should be analysed and published.

Recommendations  Finalize the identification and publish the results.

Anastrepha ludens, A. serpentina, A. obliqua and A. striata Participants: Mariana Mateos, Humberto Martinez-Montoya (USA) Collaborators: Jorge Toledo, Emilio Hernandez (Mexico)

5 year plan  Compare pyrosequencing results to culture-dependent results.  Conduct further pyrosequencing analyses of irradiated (sterile) and non –irradiated males, to evaluate the effect of irradiation on microbiota.

Achievements  The changes caused by irradiation were described and the images indicated that the midgut of male degenerates completely days after treatment with irradiation and decresed the diversity and abundance of the population bacteria.

Gaps  Identify the community bacteria of the irradiated bacteria.

Recommendations 47

 Continue with the identification of the community bacteria of the irradiated bacteria.

Anastrepha fraterculus Participant: Diego Segura (Argentina) Collaborator: Boaz Yuval (Israel)

5 year plan  Description of the main bacteria inhabiting the gut of wild flies and laboratory strains through the characterization of the 16S rDNA gene.  Assessing differences in gut bacterial community among wild caught flies, wild flies that have emerged in the laboratory and laboratory flies following the methodology described above.  Evaluation of changes in the gut microbiota due to adaptation to laboratory conditions following the methodology described above.

Achievements  The gut bacterial community was characterized in wild and laboratory males by DGGE followed by sequencing of selected bands. Several OTUs were found. The majority of the bacteria were Enterobacteria from the order Gammaproteobacteria.  Through culture dependent methods, bacteria inhabiting guts of laboratory males, wild-caught males and males emerged from wild pupae (recovered from guava fruit) were isolated and partially characterized thorugh colony PCR and sequencing. A strain collection was established for further use.  The effect of domestication of wild flies to laboratory condition was assessed through NGS. A significant effect in terms of gut bacterial diversity was detected. Aditionally, the age of the adult and the host where the larva developed had a significant effect on the composition of the bacterial community associated the the adult guts.

Gaps  Description of the gut microbiota in wild caught males through culture independent methods.

Recommendations  Consider spatio-temporal patterns in the characterization of the microbiome in different regions of the gut possibly using FISH and/or SEM.

Genus Bactrocera

Zeugodacus cucurbitae, Bactrocera dorsalis Participant: Mahfuza Khan (Bangladesh) Collaborators: Kostas Bourtzis (IAEA) and George Tsiamis (Greece)

5 year plan  Isolation and identification of the gut microbial community of laboratory (host) reared Z. cucurbitae and B. dorsalis using classical microbial and biochemical tests.  Collection four Bactrocera species (Z. cucurbitae, B. dorsalis, B. tau, and B. zonata, from four different districts of Bangladesh using cue-lure and methyl-eugenol baited

traps). Identification of gut microbial community using molecular techniques (16S rRNA amplicon sequencing).

Achievements  Isolated and identified with API strips the gut microbial community of laboratory (host) reared Z. cucurbitae and B. dorsalis using classical microbial and biochemical tests. Refer to table 4.  Collected microbiota samples for 16s rRNA community sequencing for all four species  DNA has been extracted from bacterial communities and 16S rRNA libraries have been constructed.  Data analysis is pending

Gaps  Lack of dissected material from natural populations.

Recommendations  Perform scientific sampling explorations.

Zeugodacus cucurbitae Participant: Ramesh Hire, Ashok Hadapad (India)

5 year plan  Isolation and characterization of gut microbiota of Z. cucurbitae using molecular techniques.  Phylogenetic analysis of gut microbiota.

Achievements  The gut microbiota was isolated and identified using 16S rRNA sequencing.  Citrobacter, Klebsiella, Providentia, Entobacter species were predominant.  Culture filtrates of these bacteria signficantly attracted the females and males of B. dorsalis and Z. cucurbitae.  The volatile compounds from the culture filtrates were identifed and majority of the volatiles belong to alcohols and sulpher compounds.  Majority bacteria belong to family Enterebactoraceae, Bacillaceae, Micrococcaceae and Staphylococcaceae.  The sequences of isolated gut bacteria were submitted to GenBank.

Gap identified  The contribution of individual volatile compound or a cocktail of few compounds can studied in attracting different fruit fly species.

Recommedations  The attractant potential of gut bacterial filtrates could be studied to trap the different Bactrocera species  The potenital attractants then can be used to trap females of the Bactorcera species in field.

Bactrocera dorsalis, Bactrocera minax Participant: Changying Niu (China)

5 year plan  Collect specimens from the wild population and lab colony of B. dorsalis and B. minax;  Dissect mid-gut and extract bacterial DNA.  Use second-generation DNA sequencing technology to identify the bacteria in the midgut of B. minax，study the composition, diversity of gut symbiotic microbiota through the high-throughput sequencing of bacteria 16S rDNA V6 sequence  Compare microbiota structure and diversity in B. dorsalis and B. minax.

Achievements  Identfied the diversity and structure of bacteria across life stages of B. dorsalis and B. minax by pyrosequencing analysis.  Isolated and identified the bacteria from the female and male adults of wild population in B. dorsalis by culture dependent approached.  Isolated and identified the bacteria from the adults for genetic sexing strain of B. dorsalis.  For B. minax identified the bacteria from the gut of adults, the ovipositor, the laying egg mark, and citrus flesh, which suggested vertical transmission.  Bacteria supplemented diets increased the fecundity and mating in B. minax, implying that they could improve on fitness of adults.

Gaps  Compare microbiota structure and diversity in B. dorsalis and B. minax.

Recommendations  Develop volatile substance from the specific bacteria target female flies in B. dorslis and B. minax.  Explore specific bacteria related to nitrogen fixation and immune response in B. minax larvae and adults.  Figure out the specific bacteria related to long pupal diapause in the 3rd instar larvae and pupae in B. minax.

Bactrocera oleae Participant Antonio Belcari, Patrizia Sacchetti

5 year plan  Determine the presence of Ca. Erwinia dacicola in the olive fly populations  Verify the presence of Ca. Erwinia dacicola in wild populations in different olive crop areas.  Investigations on the gut microbiome of lab reared flies.

Achievements  Bacterial DNA content of the oesophageal bulbs was analysed through PCR and endonuclease resctriction enzymes.

 The presence of the bacterial symbiont Ca. Erwinia dacicola was highlighted in the samples from all the three groups, although with a weaker signal in younger specimens and in those irradiated with 150 Gy. In these preliminary observations the presence of different bacterial species was not verified.

Gaps  None

Recommendations  None

Zeugodacus cucurbitae, Bactrocera zonata Participants: Sabrina Dyall, Preeaduth Sookar, Malini Alleck (Mauritius)

5 year plan  Collection of wild Z. cucurbitae and B. zonata adult males from 5 geographical locations in Mauritius and from laboratory-reared flies, at 3-monthly intervals  Isolation of gut microbiota and cultivation of bacteria  Extraction of DNA and PCR amplification of 16S rDNA fragments from cultivated bacteria followed by sequencing  Extraction of community DNA and PCR amplification of bacterial-specific and archaeal-specific 16S rDNA fragments, and of eukaryotic-specific markers; production of libraries from the three domains.  Test isolated bacteria on adult populations to improve longevity, male mate competitiveness

Bactrocera tryoni, Bactrocera neohumeralis, Bactrocera cacuminata Participant Olivia Reynolds, Toni Chapman, Peter Crisp, Markus Riegler, Deane Woruba, Jen Morrow (Australia)

5 year plan  Collection of B. tryoni from the Fruit Fly Production Facility (FFPF) before and after irradiation and other native and endemic Bactrocera spp. from different areas of Australia and different vegetation.  Identification of microbial communities using molecular techniques (16S rRNA sequencing). Compare microbial communities of wild B. tryoni (and other Bactrocera spp.) with both irradiated and non-irradiated communities of FFPF B. tryoni.  Isolate and identify (through molecular profiling) probiotic candidate bacteria from sampled flies.

Achievements  Characterisation of the bacteria in the gut of domesticated irradiated/fertile, teneral and mature adult B. tryoni, fed a full diet (i.e. yeast and sugar) or a sugar only diet.  Isolation and commencement of identification of microbial symbionts of male and female, mass-reared and wild B. tryoni sourced from different vegetation from several geographical locations across Australia (native range) and New Caledonia (invasive range). Gaps 51

 Identification of probiotic candidates. Recommendations  Complete the gap identified above.

Genus Ceratitis

Ceratitis capitata Participants: Hernán Donoso, Carolina Yáñez (Chile)

5 year plan  Isolation of gut microbiota and cultivation of bacteria.  DNA extraction and PCR amplification of 16S rRNA genes for DGGE analysis.  Identification of microbial communities in adults of the laboratory colony under mass rearing and small scale conditions.

Achievements  Total and diazotrophic bacterial communities from two different fly colonies (injection and release streams) were studied using DGGE and culture-dependent methods.  Bacterial strains were identified by 16S rRNA sequencing.  Similarly, the gut microbiota from wild flies was characterized. Among diazotrophs obtained from wild flies, we isolated Klebsiella sp., as found in mass-reared flies, and Kluyvera sp., not reported among isolates from mass-reared flies analyzed during this project.

Gaps  Sequence dominant bands in the DGGE gel of the gut bacterial community (total and diazotrophic) in the two fruit fly colonies and wild flies.  Compare bacterial communities in mass reared adults and wild flies by 16S rRNA amplicon sequencing.

Recommendations  16S rRNA amplicon sequencing in wild flies.  Isolation of bacteria from wild flies.  Introduction of bacteria isolated from wild flies into the adult diet.  Quality assessment of the bacterial fed adults.  Evaluation of VOCs produced by bacteria.

Participants: Jaime Garcia de Oteyza and Teresa Navarro (Spain)

5 year plan  Identification of microbial communities in adults of wild and mass reared medfly (different strains and colonies), by molecular and classical approaches (DGGE, 16S rRNA sequencing, pyrosequencing).

Achivements

 Identification of microbial communities in adults of wild and mass reared medfly (different strains and colonies), by molecular and classical approaches (DGGE, 16S rRNA sequencing).  Strain collection available.  Identification of yeasts in wild and mass reared medfly adults has been carried out by molecular approaches (ITS region).

Gaps  Evaluation of human safety / pathogenicity of the isolated strains.

Participants: Aydin Tuncbilek (Turkey)

5 year plan  Collect wild Ceratitis capitata populations from different region of Turkey and over different seasons.  Characterization of gut microbiota in wild and lab-reared flies using 16S rRNA sequencing.

Achievements  Gut microbiota in wild and lab-reared flies was isolated and identified using 16S rRNA gene sequencing.  The medfly gut bacterial communities, in culture-dependent approach were dominated by two phyla: Proteobacteria, and Firmicutes.  Enterobacteriaceae seen most commonly bacteria families present in wild and lab- reared flies.  The species richness of field-collected C. capitata seemed higher than in their corresponding laboratory samples.

Gaps  Identification of the gut bacteria by 16S rRNA gene in culture-independent approach will be included.

Recommendations  None

Genus Dirioxa

Dirioxa pornia Participant Peter Crisp (Australia)

5 year plan  Isolate bacteria from D. pornia from multiple populations and varied crops.

Achievements  The bacterial community in the entire intestinal tract of 12 individual D. pornia has been sequenced using NGS. All 12 flies were collected from the same tree on the same day.

o Of these 8 (5 female and 3 male) were of a suitable quality for analysis. o These communities appeared to differ among flies but is yet to be statistically analysed. Gaps  The mode of action of the beneficial bacteria is unknown and further elucidation is required.  Location of beneficial within the gut and changes in density and location in the gut over time is unknown, a better understanding of this will assist improve understanding of the role of the bacteria and optimal timing of supplementation.  The potential of transmission of the selected bacteria to larvae in a commercial production facility is unknown and will assist optimisation of the provision.

Recommendations  None

Summary of the Structure of Microbiota from Adults

Previously the structure of multiple tephritid species had been assessed with culture- dependent means and identification of those bacterial from 16s rDNA sequencing. The current work focuses on both lab-reared and wild populations of adult tephritids and employs the most current sequencing technology, next generation sequencing (NGS), to identify and classify bacterial OTUs. NGS methodology allows for a more complete and less biased picture of the bacterial community. During the course of the CRP, Anastrepha ludens, A. obliqua, A. serpentina, A. striata, A. fraterculus, Bactrocera cucurbita, B. dorsalis, B. minax, Ceratitis capitata and D. pornia wild and lab-reaered populations were sequenced using NGS technology. The analyses of the bacterial community are on-going, but will greatly add to the knowledge of the adult tephritid microbiome. Additionally Bactrocera oleae and B. tryoni adult bacterial microbiomes were also analysed with DGGE and 16s rDNA sequencing using Sanger methods. For two species, Anastrepha obliqua and A. serpentina, bacteria-enriched diets were tested and resulted in the identification of several bacteria with the potential for probiotic application. .

2.2. Effects of radiation

Genus Anastrepha

Anastrepha fraterculus Participant: Diego Segura (Argentina) Collaborator: Boaz Yuval (Israel)

5 year plan  Determine the effect of irradiation (X-rays) on the gut microbial community of laboratory flies through the characterization of the 16S rDNA gene. Plans for the third 18 months.  Continue the characterization of the gut bacterial community of irradiated flies by means of DGGE followed by sequencing of selected bands and analysis of DGGE profiles.

Achievements  Amplification of 16S rRNA gene (V6 region) from the gut of irradiated (70 Gy) males and females is completed.

Gaps  DGGE or other alternative approach (NGS) in order to determine the effect of irradiation on gut microbiota.

Recommendations  Effect of the irradiation on the gut microbiota of flies provided probiotics.  Physicochemical conditions of the gut after irradiation.

Anastrepha ludens, A. serpentina, A. obliqua and A. striata Participants: Mariana Mateos, Humberto Martinez-Montoya (USA) Collaborators: Jorge Toledo, Emilio Hernandez, Marysol Aceituno (Mexico)

5 year plan  Conduct pyrosequencing analyses of irradiated (sterile) and non–irradiated males, to evaluate the effect of irradiation on microbiota.

Achievements  Collected microbiota samples from irradiated and non-irradiated males.

Gaps  None

Recommendations  None

Genus Bactrocera and Zeugodacus

Zeugodacus cucurbitae, Bactrocera zonata Participant: Sabrina Dyall, Preeaduth Sookar, Malini Alleck (Mauritius)

5 year plan  To study the effect of radiation on the microflora of the alimentary tract of the fruit flies  To study the effect of irradiation on laboratory-reared Z. cucurbitae and B. zonata adult male flies: cultivate and identify, using molecular tools, bacteria from irradiated and non-irradiated flies

Zeugodacus cucurbitae, Bactrocera dorsalis Participant: Mahfuza Khan (Bangladesh) Collaborators: Kostas Bourtzis (IAEA), and George Tsiamis (Greece)

5 year plan

 Observation on the possible change of gut microbial community of laboratory reared (host/artificial larval diet) irradiated (sterile) and control male Z. cucurbitae and B. dorsalis using molecular techniques (16S rRNA amplicon sequencing).

Achievements  The microbiota of irradiated and non-irradiated Z. cucurbitae on two different diets has been characterised using an amplicon-sequencing approach.

Gaps  The effect in all developmental stages is not determined.

Recommendations  Complete the analysis on B. dorsalis.

Zeugodacus cucurbitae Participant: Ramesh Hire, Ashok Hadapad (India)

5 year plan  Study the effect of irradiation on gut microbiota of Z. cucurbitae.  Comparative analysis of microbiota before and after irradiation.

Achievements  Different doses ranging from 10-100 Gy were used to study the effect of radiation on microbiota.  Adult emergence varied from 62-85% for different doses.  Gut microbiota was not very significantly affected in the doses we have studied.  Few bacterial species which were observed before irradiation were not observed after exposure to higher doses.  Based on 16S rRNA sequencing, Enterobacter, Bacillus, Klebsiella, Citrobacter, Staphylococcus, Acinetobacter, Providencia and Kluyvera species have been identified. The comparative analysis will be further undertaken.  Klebsiella species which were observed in non-irradiated and low dose (30-40) exposed samples were not observed when higher doses (50-70 Gy) were used.  Comparative analysis of gut microbiota pre and post-irradiation was conducted using Next Generation Sequencing (NGS).  Different doses of radiation were used to study the optimum sterility dose for male and female Z. cucurbitae.

Gaps  The effect of optimised radiation doses on microbiota need to be checked to confirm the whether these doses have any detrimental effect of microbiota.  The radiation doses to assure female sterility could not be completed and needs to be taken up.

Recommendations  None

Bactrocera dorsalis Participant: Changying Niu (China)

5 year plan  Irradiate the GSS of laboratory colony for B. dorsalis male at dosage 90 Gy.  Supplement probiotics in the adult diets and determine the bacterial difference before and after irradiation.  Conduct cage experiments to investigate whether probiotics will improve the mating competitiveness and survival ability for B. dorsalis sterile males.

Achievements  The cocktail bacteria enriched diet of Citrobacter freundii and Enterecoccus phoeniculicola could improve mating performance of sterile male in genetic sexing strain (GSS) of B. dorsalis. In addition, the percentage of female remating that firstly mated with bacteria enriched sterile male would be lower. The cocktail bacteria enriched diets also shorten the mating duration of adults.

Gaps  Explore the difference of bacteria diversity before and after irradiation of GSS of B. dorsalis.

Recommendation  None

Bactrocera oleae Participant Antonio Belcari, Patrizia Sacchetti (Italy)

5 year plan  Evaluation of presence of Ca. Erwinia dacicola and/or other bacteria in the olive fly sterilized males.

Achievements  The presence of Ca. Erwinia dacicola was assessed in B. oleae adults emerged from pupae obtained from infested larvae and irradiated at Calliope plant (ENEA, La Casaccia, Roma, Italy) with 100 and 150 Gy, 4 days before the emergence of adults.  Adults from the two irradiated groups, plus the control group, have been dissected at different age: newly emerged or 5, 10 and 20 days old flies.  Bacterial DNA content of the oesophageal bulbs was analysed through PCR and endonuclease resctriction enzymes.  The presence of the bacterial symbiont Ca. Erwinia dacicola was highlighted in the samples from all the three groups, although with a weaker signal in younger specimens and in those irradiated with 150 Gy. In these preliminary observations the presence of different bacterial species was not verified.

Gaps  None

Recommendations  None

Genus Ceratitis

Ceratitis capitata Participants: Hernán Donoso, Carolina Yáñez (Chile)

5 year plan  Isolation of gut microbiota and cultivation of bacteria in irradiated adults.  DNA extraction and PCR amplification of 16S rRNA genes for DGGE analysis.  Identification of microbial communities in irradiated adults of the laboratory colony under mass rearing and small scale conditions.

Achievements

 Gut bacterial strains were obtained from irradiated and fertiles males (injection and release streams). Identification was done by 16S rRNA sequencing.  Gut bacterial community was characterized by DGGE. Changes in diversity and richness were observed.

Gaps  Compare bacterial communities in irradiated and fertile mass-reared adults by 16S rRNA amplicon sequencing.

Recommendations  Bacteria are not killed by radiation, but changes in bacterial communities are observed. Studies should be conducted in order to determine the real effect of the radiation.

Participants: Jamie Garcia de Oteiza Teresa Navarro (Spain)

5 year plan  Isolation of gut microbiota and cultivation of bacteria in irradiated adults.  DNA extraction and PCR amplification of 16S rRNA genes for DGGE analysis and next generation sequencing.  Identification of microbial communities in irradiated adults of the laboratory colony under mass rearing and small scale conditions.

Achievements  Tested effects of probiotic supplementation in irradiated males.

Gaps  Need to determine best time to offer the probiotic during rearing.  Identification of yeast for probiotic use.

Participants: Aydin Tuncbilek (Turkey)

5 year plan  Collect wild Ceratitis capitata populations from different region of Turkey and over different seasons.  Characterization of gut microbiota in irradiated lab-reared flies using 16S rRNA sequencing.

Achievements

 Gut microbiota in wild and lab-reared flies was isolated and identified using 16S rRNA gene sequencing.  The medfly gut bacterial communities, in culture-dependent approach were dominated by two phyla: Proteobacteria, and Firmicutes.  Enterobacteriaceae seen most commonly bacteria families present in the gut of both irradiated and unirradiated adults.  The radiation procedure did not influence the gut bacterial structure of the wild and laboratory medfly samples.

Gaps  Identification of the gut bacteria by 16S rRNA gene in culture-independent approach will be included.

Recommendations  None

Summary of the Effects of Radiation

The two measures of radiation effects studied were comparisons of the microbiomes in non- irradiated and irradiated flies and the addition of probiotics to adult diets fed to irradiated males. Previous to the CRP there was very little information about the effects of radiation on microbiota. Using a variety of molecular techniques including DGGE, 16s rDNA amplicon sequencing and NGS, the effects on the microbiota were evaluated for Anastrepha fraterculus, Bactrocera cucurbitae, B. oleae and Ceratits capitata. The second measure identified probiotic supplementation that improved the status of irradiated males. Probiotic enriched diets were evaluated for two species, B. dorsalis and C. capitata.

2.2. Function of microbiota and fitness effects

Genus Anastrepha

Anastrepha fraterculus Participant: Diego Segura (Argentina) Collaborator Boaz Yuval (Israel)

5 year plan

 Evaluate the effect of gut bacteria on mating competitiveness, by assessing the mating choice of A. fraterculus females facing males treated with antibiotics and untreated males under laboratory conditions. Fly origin (laboratory and wild) and nutritional status (protein-fed and protein-deprived) dependent responses will be assessed.  Evaluate the effect of gut bacteria on male sex pheromone and cuticle hydrocarbons (CHC). Differences in sex pheromone and CHC between antibiotic treated males and non-treated males will be assessed by GC-FID/GC-MS.  Evaluate the effect of gut bacteria on starvation resistance by comparing (under laboratory conditions) survival rates of males treated with antibiotics and untreated males. Fly origin (laboratory and wild) and nutritional status (protein-fed and proteindeprived) dependent responses will be assessed.  Evaluate the impact of antibiotics on flies’ ingestion rate, nutritional reserves and adult dry weight.  Evaluate the effect of adding bacteria to the adult diet on male mating competitiveness and on female fecundity.

Achievements  The effect of antibiotic treatment on mating competitiveness, calling behavior, pheromone release, CHC profiles, nutritional status, starvation endurance, ingestion rate and adult dry weight was determined both for wild and laboratory males. For wild males, starvation endurance was negatively affected by antibiotic for sugar fed males. For laboratory males, antibiotics negatively affected mating competitiveness, pheromone release rate, calling behavior, and protein reserves. On the other hand, antibiotics positively affected starvation resitance and lipid reserves, but only in sugar fed males.  The effect of adding Enterobacter (two strains, one isolated from wild males and one isolated from lab males) to the larval diet on the duration of the larval stage, egg to pupae recovery and pupal weight was assessed. No significant effects were found.

Gaps  Evaluate the effect of adding bacteria to the larval and/or adult diet on reproductive parameters and starvation endurance.

Recommendations  Harmonized methods to test probiotics effects.  Define what can be considered an improvement in terms of mass rearing and performance of released sterile flies.

Genus Bactrocera

Zeugodacus cucurbitae, Bactrocera zonata Participant Sabrina Dyall, Preeaduth Sookar, Malini Alleck (Mauritius)

5 year plan  To investigate the effect of isolated bacterial symbionts of Z. cucurbitae and B. zonata during pre-release period on sterile insect performance.

Bactrocera oleae Participants: Boaz Yuval, Edouard Jurkevitch, Inbar Shuster-Dagan, Michael Ben-Yosef, Doron Zaada (Israel)

5 year plan  For Bactrocera oleae, determine the bacterial contribution to olive fly fitness in relation to its nutritional ecology.  Determine how bacteria enable the olive fly to develop in olives.

Achievements  Bacteria counter-acted the effect of oleuropein on larvae.  Determined activity of oleuropein in olives as reducing lysine Gaps  Determing the molecular mechanism whereby the olive fly symbiont enables development in unripe olives  The inability to grow Ca. Erwinia dacicola, the olive fly symbiont, in culture, severely hampers research efforts. Future work should tackle this difficult problem.

Recommendations  Investigate the utility of perpetuating the symbiont of wild flies in laboratory colonies. Alternatively, identify other bacterial species that could replace this symbiont in artificial environments. This could lead to economically viable methods for mass rearing this insect.

Bactrocera dorsalis and Bactrocera minax Participant: Changying Niu (China)

5 year plan  Supplement probiotics in the diets for B. dorsalis and B. minax.  Compare the survival, weight, pupation rate of larvae with control.  Study the fitness effects for B. dorsalis.

Achievements  The addition of the probiotic Klebsiella oxytoca found to significantly increase the average mating frequency, life span and eggs laid by B. minax.  Based on the formulated Q diets, the pH 3.0 of propionic acid, 23℃ with dark environment, and supplement of Citrobacter freundii, together contribute to improve the hatching rate of eggs of B. minax significantly.  The addition of the probiotic Klebsiella oxytoca found to significantly increase the average mating frequency, life span and eggs laid by B. minax.

Gaps  Test more probiotic bacteria to improve adult fitness in B. minax.

Recommendations  None

Genus Ceratitis

Ceratitis capitata Participant Peter Crisp (Australia)

5 year plan  Commence feeding studies using individual bacteria and assess probiotics effect on longevity and quality of irradiated male C. capitata.  Elucidate the behavioural changes in C. capitata induced by exposure to volatile compounds identified from the bacteria.

Achievements  Adult D. pornia, Ceratitis capitata and Bactrocera tryoni were exposed to agar plates inoculated with bacterial isolates to assess attraction of the flies to the bacteria. (Cultures were 3, 50+ and 100+ generations domesticated respectively).  Three of the bacteria (Enterobacter aerogenes, Enterobacter sp. and Citrobacter freundii) were highly attractive to all three species when supplied as a dietary supplement on agar.  When provided to irradiated C. capitata in the 48 hours after eclosion the treated flies were observed to be more active and dispersed more quickly than flies supplied with agar and sugar. Gaps  The mechanism that results to changes in the bacterial community during and after irradiation are uncertain. Recommendations  Further investigate radiation damage and the potential of post irradiation pro-biotics to restore structures and organs with 3D imaging.

Participants: Jaime Garcia de Oteyza, Teresa Navarro (Spain)

5 year plan  Identify suitable beneficial bacteria and quality control after their inoculation (longevity under stress, flight capacity, eggs/female…).

Achivements  Identified bacteria with 16s rRNA sequence analysis. Refer to table 4.  Develop procedures for probiotic delivery under mass rearing conditions.

Gaps  None

Recommendations  None

Participant Nikos Papadopoulos (Greece)

Collaborators: K. Bourtzis (IAEA) and G. Tsiamis (Greece)

5 year plan  Effects of probiotic provision of symbionts on the sexual behaviour of Vienna 8 males.

Achievements  Effects of probiotic symbionts on demographic and quality traits of the Vienna 8 strain have been determined.  Effects of probiotic provision of symbionts on the sexual behaviour of Vienna 8 males have been determined in the laboratory of FAO/IAEA in Siebersdorf.

Gaps  Field releases of flies produced with symbiont diets have not been conducted.  Effect of irradiation on flies produced on symbiont enriched diets should be studied.

Recommendations  Pilot small scale SIT tests using males produced in symbiont enriched diets.

Genus Dirioxa

Dirioxa pornia Participant Peter Crisp (Australia)

5 year plan  Feeding studies to assess longevity, fecundity and quality of D. pornia continued.  Continue lure and kill trials with D. pornia.

Achievements  Adult D. pornia, Ceratitis capitata and Bactrocera tryoni were exposed to agar plates inoculated with bacterial isolates to assess attraction of the flies to the bacteria. (Cultures were 3, 50+ and 100+ generations domesticated respectively).  Three of the bacteria (Enterobacter aerogenes, Enterobacter sp. and Citrobacter freundii) were highly attractive to all three species when supplied as a dietary supplement on agar.  When supplied as a dietary supplement at eclosion both Enterobacter sp. were found to significantly increase the fecundity of female D. pornia and B. tryoni and resulted in increased next generation adult eclosions.  Another of the bacteria isolates (Bacillus amyloliquefaciens) significantly delayed egg laying in D. pornia and reduced total fecundity by greater than 60%. Gaps  The mode of action of the beneficial bacteria is unknown and further elucidation is required.  Location of beneficial bacteria within the gut and changes in density and location in the gut over time is unknown, a better understanding of this will assist improve understanding of the role of the bacteria and optimal timing of supplementation.

 The potential of transmission of the selected bacteria to larvae in a commercial production facility is unknown and will assist optimisation of the provision. Recommendations  None

Summary of Microbiota and Fitness Effects

In the previous sections, the addition of a probiotic enriched diet increased the general fitness of adult Anastrepha obliqua (adult survivorship and mating performance) and A. serpentine (adult survivorship) and irradiated males of Bactrocera dorsalis (mating performance) and Ceratitis capitata. In addition to adult survirvorship and mating performance, multiple fitness measures were also evaluated.

The effects of antibiotics on fitness were analysed in A. fraterculus. Specifically, male competitiveness, pheromone release, calling behaviour, nutritional status, starvation endurance and adult weight were compared between adults exposed to antibiotics and no exposure as well as diet type. The effects of a probiotic diet were measured in B. minax adults for average mating frequency, life span, eggs laid and hatch rate. Non-irradiated C. capitata adults were evaluated for activity when exposed to bacterial attractants and the demographic effects of endosymbionts. D. pornia adults were also exposed bacterial attractants and evaluated for activity. The fitness effects of the microbiota varied from no effect to a positive effect for the different tephritid species and bacterial probiotic/antibiotic tested.

2.3. Applications

Genus Anastrepha

Anastrepha ludens Participants: Erin Schuenzel, Hugh Conway, Basilio Salas, Don Vacek (USA)

5 year plan  Track presence of probiotics given to Anastrepha ludens larvae in the adult midgut using marked bacteria and selective media.  Genotype probiotic strains surviving in A. ludens adults using MLST or newly developed genetic markers.  Assess ability to produce probiotic strain for large scale application.  Make strains available for adult fitness tests.

Achievements  No beneficial bacteria were identified with the small cup tests for the over 400 isolates tested. Gaps  Beneficial bacteria may work as a community, therefore the testing methods may not be adequate for identifying beneficial bacteria.

Recommendations  None

Participants: Dyna Melgar (Guatemala)

5 year plan  Isolation and identification through molecular techniques RT- PCR (16S rRNA) of entomopatogenic and endosymbiotic bacteria for both wild and mass reared insects.  Evaluation of the effect of endosymbiotic bacteria inoculation in the adult diets in mass rearing facilities.  Endosymbionts inoculation in agar diets and pellum-cellulose matrix in release centre and measure impact on insect fitness.

Achievements  Microbial profile characterization in egg suspension by biochemical test.  Beneficial bacteria characterization (Klebsiella sp., Enterobacter sp. and Raoultella sp.).  Pathogenic bacteria characterization (Morganella sp., Providencia sp. and Serratia sp.).  Protocol to introduce probiotics in mass rearing facility.  Addition of Raoultella ornithinolitica in the egg’s suspension.  Microbial profile characterization of liquid diet.

Gaps  Isolation and identification through molecular techniques RT- PCR (16S rDNA) of entomopatogenic and endosymbiotic bacteria for both wild and mass reared insects is still not completed.

Recommendations  Data base collection of microorganism with the beneficial and pathogenic effects in different fruit fly species.  Development of general protocols for beneficial bacteria introduction/applying in mass rearing and release facilities.  Introduction of wild beneficial bacteria in mass rearing facilities and release.  Genome sequencing of pathogenic and beneficial bacteria.  Typification of microorganisms when the mass rearing insect strain is refreshed with wild material.  Development of international shipment protocols in order to send biological material, including the microbiota.  Assess the inoculation of purposely grown endosymbionts to favor/preserve the quality of eggs during the shipment.

Anastrepha fraterculus Participant: Diego Segura (Argentina) Collaborator Boaz Yuval (Israel)

5 year plan

 Evaluation of methods to restoring bacteria identified in laboratory and wild males kept under specific dietary treatments into the sterile laboratory male gut.  Evaluation of the effect of restoring key bacteria on sterile male sexual competitiveness, male sex pheromone and CHC, and survival.

Achievements

 154 isolates have been obtained from laboratory and wild males. Bacteria from the genera Enterobacter, Klebsiella, Enterococcus, Erwinia and Citrobacter have been detected.

Gaps  Evaluation of bacterial isolates on parameters associated to the adult stage in sterile males.

Recommendations  Development of standard procedures to assess the effect of bacteria on larval, pupal and adult performance.

Genus Bactrocera

Zeugodacus cucurbitae, Bactrocera zonata Participants: Sabrina Dyall, Preeaduth Sookar, Malini Alleck (Mauritius)

5 year plan  To design probiotic diets that will improve fitness of B. cucurbitae and B. zonata irradiated adult males  To determine the effects of the probiotic diets on the following quality control parameters: egg hatch, pupal weight, calling and mating behaviour

Zeugodacus cucurbitae, Bactrocera dorsalis Participant Mahfuza Khan (Bangladesh)

5 year plan  Semi-field cage experiments on the effect of probiotic adult diets as pre-release supplement to enhance the mating competitiveness of control and sterile Z. cucurbitae and B. dorsalis.  Determine the survival of control and sterile Z. cucurbitae and B. dorsalis fed on probiotic adult diets under semi-field cage trials.  Test the attraction of adult Z. cucurbitae and B. dorsalis to different gut bacteria species.

Achievements  Determined the effect of gut bacteria enriched diets on mating, fecundity and survival under laboratory and semi-field conditions.

Gaps  Further semi-field cage experiment should be conducted using sterile flies to confirm the lab findings with gut bacterial spp. on mating succsses longevity, and fecundity of Z. cucurbitae and B. dorsalis.

Recommendations  Use of Klebsiella oxytoca and Providencia rettgeri into protein and sugar diets may be used to enhance the mating success of sterile B. dorsalis.  The effect of bacteria on different fitness parameters is diet-dependent. Use of gut bacterial isolates in protein diets improves longevity and fecundity of Z. cucurbitae and B. dorsalis.  Adding of some gut bacteria spp. in sugar diet may increase the mortality of Z. cucurbitae and B. dorsalis. Whereas, some gut bacteria species Erwinia sp., Serratia spp. have indirect effect and helps to delay egg development of Z. cucurbitae and B. dorsalis while added with sugar diet.

Bactrocera oleae Participant Antonio Belcari, Patrizia Sacchetti (Italy)

5 year plan  Development of bacterial symbiosis transfer (Ca. Erwinia dacicola from wild flies to lab reared flies).  Development of a probiotic diet for the adult.

Achievements  Probiotic diets based on two bacterial species, P. putida and A. tropicalis, were administered to lab reared olive fly at two quantity level (one the double of the other) and at two different status (alive and heat killed). Only few probiotic diets showed general positive effects on adult olive flies, in general no one treatment produced consistent effects on physiological fly parameters. No consistent differences have been evidenced between live and heat-killed bacteria nor between suspension concentrations, although the bacterial dose influenced some parameters.  It was confirmed that the bacterial symbiont Candidatus Erwinia dacicola is consistently present in wild olive fly populations while it was never evidenced in lab reared olive flies by the investigations carried out during the CRP. Possibly, lab reared colonies can establish symbiotic relationships with different bacterial species favoured by lab conditions.  The transmission of the symbiont Ca. Erwinia dacicola from olive fly female to the progeny via eggs was proved using molecular techniques and electron microscope. Laboratory experiments using wild olive flies, highlighted that the symbiont transmission can be hampered by washing eggs with disinfectants or mild antifungal agents like propionic acid, commonly used to prevent fungal growth in olive fly larval diet.  Lab trials aimed at development of symbiont transmission from wild olive fly populations to lab reared colonies have been designed and carried out. Lab reared olive flies adults were kept in contact for 14 days with different substrates, possible sources of the bacterial symbiont Ca. E. dacicola. After that flies were dissected, bacterial DNA content of the oesophageal bulbs was extracted and amplified samples

were analysed through DGGE using specific primers. Then the species was confirmed by gene sequencing.

Gaps  Probiotic diets administration techniques should be improved. Difficulties also in assuring all the tested insects fed on probiotic diet.

Recommendations  Have better quality standards when choosing proper primers and possibly to perform different analyses on the same sample.

Bactrocera dorsalis Participant: Changying Niu (China)

5 year plan  Clone the full-length genes related to spermatogenesis of male B. dorsalis.  Study the function of the selected genes by RNAi in B. dorsalis.  Express dsRNA of genes in E. coli strain HT-115.  Feed these bacteria to the larvae of B. dorsalis in the diets.  Conduct lab and cage experiments to examine mating, oviposition of females, and hatching rate of the offspring.

Achievements  We use RNAi to study the function of spermatogenesis related genes in male of B. dorsalis, which suggested that the knockdown rho gene could significantly affect the fecundity and fertility. We recorded a significant decrease for the number and length of sperms in female spermatheca in dsrho treated group. In a greenhouse trial, the number of damaged oranges and B. dorsalis larvae were significantly reduced in a dsrho-treated group compared with the dsgfp control group. This study provides strong evidence for the use of RNAi in pest management, especially for the improvement of SIT against B. dorsalis and other species.

Gaps  Investigate the prevalence of Wolbachia in different populations of B. dorsalis and B. minax.

Recommendations  Based on the available genome information of fruit fly species, study the key gene function related to visual, olfactory, tactile of adults by RNAi technology, the goal is to improve SIT application in the long run.

Bactrocera tryoni Participant Olivia Reynolds, Toni Chapman, Peter Crisp, Deane Woruba (Australia)

5 year plan  Laboratory and field cage trials assessing performance and fitness traits including longevity and mating success of bacterial supplemented flies. 68

Achievements  Testing adult probiotic candidates on B. tryoni pending.

Gaps  Influence of feeding adult B. tryoni selected probiotic candidates, on quality traits  Understanding the functional role of bacteria in adult B. tryoni. Recommendations  Complete the gaps identified above and pursue potential for utilisation of adult probiotic within SIT programs.

Genus Ceratitis

Ceratitis capitata Participants: Dyna Melgar (Guatemala)

5 year plan  Isolation and identification through molecular techniques RT- PCR (16S rDNA) of entomopatogenic and endosymbiotic bacteria for both wild and mass reared insects.  Evaluation of the effect of endosymbiotic bacteria inoculation in the adult diets in mass rearing facilities.  Endosymbionts inoculation in agar diets and pellum-cellulose matrix in release centre and measure impact on insect fitness.

Achievements  Typification of microorganisms at Emergence and Release Facilities that use genetic sexing strains of Ceratitis capitata.  Microbial profile characterization in egg suspension by biochemical test.  Beneficial bacteria characterization (Klebsiella sp., Enterobacter sp.).  Pathogenic bacteria characterization (Morganella sp., Providencia sp. and Serratia sp.).  Protocol to introduce probiotics in mass rearing facility.

Gaps  Isolation and identification through molecular techniques RT- PCR (16S rDNA) of entomopatogenic and endosymbiotic bacteria for both wild and mass reared insects is still not completed. Recommendations  Data base collection of microorganism with the beneficial and pathogenic effects in different fruit fly species.  Development of general protocols for beneficial bacteria introduction/applying in mass rearing and release facilities.  Introduction of wild beneficial bacteria in mass rearing facilities and release.  Genome sequencing of pathogenic and beneficial bacteria.

 Typification of microorganisms when the mass rearing insect strain is refreshed with wild material.  Development of international shipment protocols in order to send biological material, including the microbiota.  Assess the inoculation of purposely grown endosymbionts to favor/preserve the quality of eggs during the shipment. Participants: Peter Crisp (Australia)

5 year plan  Establish trials to assess effect of exposure to bacterial volatiles for SIT C. capitata.  Establish field trials to assess effect of bacterial feed at emergence for SIT C. capitata.  Establish field trials to assess effect of exposure to bacterial volatiles isolated from D. pornia for SIT C. capitata.

Achievements  Preliminary collection and analysis of volatile compounds was completed using Enterobacter aerogenes and Citrobacter freundii. Samples were run against alcohol standards and 5 compounds (4 methylbutanols and a methyl acetate) were identified. Purified samples of these compounds were assessed for attractiveness to D. pornia and B. tryoni in Y-tube trials but were not significantly more attractive than water.

Gaps  The identification was limited by the standard samples available (alcohols), therefore amines and quarternery amoniums need to be elucidated and then assessed for attraction.  Alcohols were only tested in pure form and require assessment at greatly reduced concentrations.

Recommendations  Identify the volatile compounds from bacteria that are attractive to fruit flies adults, particularly females, with the aim of developing new lures. Improved trapping will enable improved tailoring of release rates and timing of SIT programs.

Participants: Jaime Garcia de Oteyza, Teresa Navarro (Spain)

Five years plan  Development of probiotic culture and supply/inoculation methodology for mass rearing programs.

Achievements  Suspected probiotic (Klebsiella oxytoca) added to adult diet did not show any benefit on sterile male QC parameters.

Gaps  None

Recommendations  Continue to try different bacteria for probiotic effect.

Participants: Spyridon Ntougias (Greece)

5 year plan

 Examine the growth characteristics and the biokinetic properties of the medfly gut symbionts Enterobacter sp. AA26 and Providencia sp. AA1 in both batch cultures and bioreactor systems for mass rearing and sterile insect technique applications.

Achievements

 Determined specific growth rates of Enterobacter sp. AA26 and Providencia sp. AA1 in batch cultures using LB as the growth medium, i.e. 2.08 and 2.19 h-1, respectively.  The pH, salt and temperature ranges for growth of Enterobacter sp. AA26 and Providencia sp. AA1 were identified.  For strain AA26: pH range, 4-10.2; salt range, 0-10% w/v NaCl; temperature range, 4-40oC.  For strain AA1: pH range, 4-9.4; salt range, 0-10% w/v NaCl, temperature range: 6-42oC.  Both bacterial isolates were capable of growing in the presence and absence of oxygen. They were oxidase-negative and catalase-positive, giving strong catalase reactions.  Therefore, strain AA26 is a psychrotolerant, halotolerant and neutrophilic (with broad pH range for growth) facultatively anaerobic bacterium, and strain AA1 is a mesophilic, halotolerant and neutrophilic (with broad pH range for growth) facultatively anaerobic bacterium.  The biochemical profiles of Enterobacter sp. AA26 and Providencia sp. AA1 were identified. As a member of the genus Enterobacter, strain AA26 can ferment lactose and glucose, producing gas, and it is oxidase-negative, indole-negative and Voges- Proskauer-positive. Moreover, it decarboxylates ornithine, lacks phenylalanine deaminase and it does not produce H2S. Providencia sp. AA1 can ferment glucose, without producing gas, and produce acetoin. It can also hydrolyze urea and utilize citrate.  The protein content of the dry biomass at the late exponential phase was calculated for both Enterobacter sp. AA26 and Providencia sp. AA1, which was equal to 56.6 ± 6.3% and 53.2 ± 3.2%, respectively (n=3). The biomass was also estimated per liter of LB medium at the late exponential phase as 2145 ± 56 and 1850 ± 30 mg dry weight for Enterobacter sp. AA26 and Providencia sp. AA1, respectively (n=3).  The biokinetic properties of the medfly gut symbionts Enterobacter sp. AA26 and Providencia sp. AA1were determined in bioreactor systems operating aseptically under the draw and fill mode. The biomass characteristics of strain AA26 were: i.e. -1 o μmax = 0.069 h 35 C, YH = 0.58 ± 0.03 g biomass/g substrate consumed, OUR = -1 -1 -1 -1 79.58 ± 1.59 mg O2 L h and SOUR= 63.16 ± 1.26 mg O2 g VSS h . The biomass -1 o characteristics of strain AA1 were: μmax = 0.067 h at 35 C, YH = 0.73 ± 0.01 g -1 -1 biomass/g substrate consumed, OUR = 43.56 ± 1.94 mg O2 L h and SOUR= 38.89 ± -1 -1 1.73 mg O2 g VSS h .  The viability of Enterobacter sp. AA26 and Providencia sp. AA1 was assessed. Both strains were viable for long period of time, i.e. more than 9 months at 6oC.

 Based on phylogenetic analysis, isolate AA26 is an Enterobacter cloacae strain, whereas isolate AA1 was related to Providencia vermicola and P. rettgeri. The positive urea hydrolysis reaction indicates that strain AA1 is a P. rettgeri strain.  The glucosidase, protease and lipase activities of Enterobacter sp. AA26 and Providencia sp. AA1 growing in both LB and cheese whey wastewater were assessed. Greater - and β-glucosidase activities where observed during growth of the strain AA26 in cheese whey wastewater, while the respective activities were almost half in the case of growth in LB broth (p<0.01). In comparison to cheese whey wastewater in which only low extracellular glucosidase activities were detected, significant higher extracellular enzyme activities were observed when strain AA26 grew in LB medium (p<0.01). Greater endocellular -glucosidase activity was determined during growth of the strain AA1 in LB than in cheese whey wastewater (p<0.01).  No pectinase activity was detected for Enterobacter sp. AA26 and Providencia sp. AA1.  The use of the anti-foaming agent in a negligible amount (20 g/L) minimized the formation of the foam during the growth of Enterobacter sp. AA26 in LB medium. PAC and polyelectrolyte concentrations of 2000 mg/L and 2 mg/L, or 3000 mg/L PAC and polyelectrolyte concentration of 1 mg/L (or above) reduced OD600 nm and SVI values below 0.06 and 265 mL/g, respectively. The investigation of FeCl3 as coagulant revealed that the optimum dose application was 1000 mg/L.

Gaps  None

Recommendations  Indeed, Enterobacter sp. AA26 and Providencia sp. AA1 were capable of growing in agricultural wastewaters (i.e. cheese whey wastewater) and therefore, use of such organic substrates can replace the high cost substrates used for the preparation of commercial available media. Moreover, extended biomass production is needed for biotechnological applications of these strains and scale-up approaches are recommended.

Summary of Applications of Microbiota

Of the multiple strains tested across several tephritid species, only several were found to have probiotic effect. For Anastrepha ludens, Klebsiella sp., Enterobacter sp. and Raoultella sp. were identified as possible probiotics. Potential probiotics were also identified for A. obliqua (Providencia rettgeri, Lactobacillus rhamnosus) and A. serpentina (Pantoea aglomerans, Providencia rettgeri, and Chryseobacterium indologenes), Bactrocera cucurbitae (Erwinia sp. and Serratia sp.), B. dorsalis (Klebsiella oxytoca and Providencia rettgeri), B. oleae (Psuedomonas putida and Acetobacter tropicalis) and Ceratitis capitate (Klebsiella and Enterobacter). In addition to diet additives, the role of bacteria as attractants was also assessed for C. capitata (Enterobacter aerogenes, Enterobacter sp. and Citrobacter fruendi) and D. pornia (Enterobacter aerogenes, Enterobacter sp. and Citrobacter fruendi). The potential of bacteria as attractants and the identification of the volatiles released by bacteria remains an area of needed study for other tephritid species.

3 SYMBIONTS AND NOVEL CONTROL TOOLS

Background situation analysis Certain symbiotic bacteria are known to manipulate the mating behaviour and reproduction of their hosts. After detecting and characterizing the occurrence of Wolbachia and other heritable symbionts in several Bactrocera and Anastrepha species there is a need to determine their phenotypes and their fitness effects on hosts, and their interactions with other associated organisms. This determination is essential to develop approaches that will allow introduction of symbionts such as Wolbachia and others into target populations with potential to effectively reduce pest populations and their economic impact. There are two potential approaches. First, the incompatible insect technique (IIT) employs cytoplasmic incompatibility, which is induced by insect symbionts such as Wolbachia. In a Wolbachia-based IIT strategy, female sterility is artificially sustained in pest populations by repeated releases of cytoplasmically incompatible mass-reared males. Since Wolbachia is not paternally transmitted, the infection type present in the release strain does not become established in the field. For this reason, IIT requires the release of males only, thus the availability of an efficient sexing technique. Similar to the conventional SIT, the increasing ratio of incompatible matings over time can lead to population suppression. Secondly, Wolbachia and other reproductive symbionts could be used to manipulate host population fitness (a) to reduce/block the capacity to transmit pathogens such as viruses through life span reduction and interference with pathogens, (b) to modulate the behaviour (feeding and mating behaviour) and (c) to impact abiotic stress resistance (thermotolerance, desiccation resistance, dormancy). Some of these approaches have already been tested in the laboratory and in the field, e.g. pathogen transmitting mosquitoes. It is worthwhile to explore their potential application against tephritid pests.

Subtheme A: Detection, molecular and phenotypic characterization

Current knowledge:

A1. General: A range of reproductive symbionts has been found in Tephritids. So far, Wolbachia seems to be the most dominant, with detection in several species of Rhagoletis, Bactrocera, Anastrepha, and Dacus. However, Wolbachia is absent from C. capitata, B. oleae, and A. ludens. Furthermore, other reproductive symbionts have been detected. The largest knowledge so far is available for Wolbachia, with a wide diversity of strains of mostly supergroup A and some of the supergroup B. Wolbachia infections occur as single or as multiple infections in individuals, with dominant and less dominant strains. The starting point of detection for reproductive parasites is the conserved bacterial 16S rDNA sequence analysis; however, this approach is not sufficient for discriminating strains that can induce different phenotypes and/or have different origins. The molecular characterization of these symbionts is made available through Multi Locus Sequence Typing (MLST) systems that have recently been developed for Wolbachia while it is only partially developed for Arsenophonus and Spiroplasma.

So far the only characterized phenotype of Wolbachia in tephritids is induction of cytoplasmic incompatibility (CI). Wolbachia infections have been reported to have either positive or negative effects on fitness of host populations. Artificially Wolbachia infected medfly lines suffer from reduced fitness such as survival and reproduction. In addition, some of the fruit flies under study are cryptic species complexes. Accurate taxonomic identification of the fly specimens used for Wolbachia detection should be guaranteed in order to establish possible associations between Wolbachia strains and particular fruit fly species/populations.

A2 Genus level background: Anastrepha: Wolbachia detected in some populations of A. serpentina, A. striata, A. obliqua, A. fraterculus, A. amita, A. sororcula, A. pickelii but not in A. ludens. With exception of A. striata (carries both A and B supergroups) all other species carry Wolbachia strains that belong to supergroup A. Bactrocera: Wolbachia detected in populations of B. dorsalis, B. philippinensis, B. carambolae, B. correcta, B. tau, B. cucurbitae, B. zonata, B. scutellaris, B. nigrofemoralis and several Australian Bactrocera species (e.g. B. neohumeralis). The prevalence of Wolbachia infection on Bactrocera species varies among and within countries. Ceratitis: Although Wolbachia has been reported in a laboratory strain in Brazil and one French population of C. capitata, recent thorough surveys of natural populations demonstrate that Wolbachia is absent in Brazil and Turkey. Dacus: Wolbachia detected in D. destillatoria from Thailand and in some Australian Dacus species. Rhagoletis: Wolbachia detected in all populations of R. cerasi, in some populations of R. pomonella, R. cingulata and R. completa. There are several Wolbachia strains present in field populations of R. cerasi. CI is expressed in field populations of R. cerasi.

Gaps identified

A1. General: (i) molecular detection  Lack of universal, cost effective, system to detect reproductive parasites.  Application of sensitive methods to detect low titer and additional strains of Wolbachia.

(ii) molecular characterization  Lack of MLST systems for reproductive parasites other than Wolbachia and Spiroplasma  Although substantial progress has been made over the last five years, there is still need for additional characterisation of symbionts in different species, populations and geographic areas

(iii) phenotypic characterization

 Despite the substantial progress made over the last five years, there is still need for additional phenotypic characterization of the interactions among symbionts  Knowledge about the effects of reproductive symbionts on behavioural traits is restricted to medfly.  Lack of understanding of the molecular mechanism causing full or partial CI.  Lack of understanding of host symbiont interactions leading to sustainable artificially infected insect pest lines. (iv) Trans-infection technology  Lack of efficient methodology to transfer Wolbachia across fruit fly species.

A2. Genus level background: Anastrepha:  There is no evidence for CI in Anastrepha species.  Knowledge of infection frequencies and types in field populations is restricted to few species and/or regions.  There is no information regarding effects of Wolbachia on host fitness.

Bactrocera:  There is no evidence for CI in Bactrocera species other than transinfected B. oleae.  Knowledge of infection frequencies and types in field populations have been advanced but there is still need for data regarding additional species and/or regions.  There are no studies on fitness effects other than CI.

Ceratitis:  Lack of compatibility studies between trans-infected Vienna 8 lines and different wild medfly populations preferably obtained from different geographic areas.  Lack of feasibility studies regarding the application of IIT.  Lack of data regarding Wolbachia presence in other Ceratitis species than medfly.

Dacus:  Limited knowledge of infection frequencies and types in field populations.  No information regarding fitness effects or phenotypes in naturally Wolbachia infected populations

Rhagoletis:  Lack of knowledge of infection frequencies and types in field populations of Rhagoletis species other than R. cerasi.  Need to continue monitoring dynamics of Wolbachia infected R. cerasi populations.  A substantial amount of data has been accumulated regarding the demographic profile of naturally infected R. cerasi population. No understanding of effects on behavioural and other than demographic biological traits in populations naturally infected by Wolbachia.  No comprehensive data exist regading quantitive aspects of Wolbachia infection in larvae, pupae and adults of different ages of R. cerasi

Subtheme B: Interactions of reproductive symbionts and other microorganisms

Current knowledge:

B1. General: The interactions between reproductive symbionts and other microorganisms are largely unknown. A good understanding of the host microbiome is required to be able to determine interactions with reproductive microorganisms. Preliminary data indicate that in artificially Wolbachia infected C. capitata the bacterial community is largely suppressed. In other insect groups such as Drosophila and mosquitoes, Wolbachia suppresses viral infection of the host, and pathogen transmission (bacteria, plasmodia, filarial nematodes, viruses).

B2. Genus level background: Anastrepha: Interactions have not been studied. Bactrocera: An artificially Wolbachia infected line has been established for the olive fly only. Interactions have not been studied. Ceratitis: Preliminary data indicate that in two artificially Wolbachia infected C. capitata lines the bacterial community is largely suppressed. Dacus: Interactions have not been studied. Rhagoletis: Interactions have not been studied.

Gaps identified:

B1. General: Characterization of the host microbiome is prerequisite to be able to determine effects of reproductive parasites. There is need to establish artificially reproductive symbiont infected lines and/or to identify infected and uninfected individuals of the same wild population in order to determine interactions.

B2. Genus level background: Anastrepha:  No artificially infected lines have been established besides some first efforts for A. ludens.  The status of naturally infected populations by reproductive symbionts has been characterized for some species. Additional data are needed for other species and populations. Bactrocera:  No artificially infected lines have been established (except B. oleae).  The status of naturally infected populations by reproductive symbionts has been characterized for some species. Additional data are needed for other species and populations. Ceratitis:  The status of naturally infected populations by reproductive symbionts other than Wolbachia has not been fully characterized. Dacus:  No artificially infected lines have been established  The status of naturally infected populations by reproductive symbionts has not been fully characterized. Rhagoletis:  No artificially infected lines have been established 76

 The status of naturally infected populations by reproductive symbionts other than Wolbachia has not been fully characterized.

Subtheme C: Applications (CI and host fitness manipulations)

Current knowledge:

C1. General: The intracellular symbiont Wolbachia can manipulate the reproductive performance of its insect hosts and among others, cause cytoplasmic incompatibility in crosses between infected males and non-infected females (Uni-directional CI), and between individuals infected with incompatible Wolbachia strains (Bi- directional CI). Wolbachia infection may be harnessed to induce CI into wild insect populations directly suppressing pest populations in an environmental friendly manner. This method, “Incompatible Insect Technique” (IIT), is similar to SIT, the only difference being the sterilization mode – IIT does not use γ-rays – and can be complementary to SIT. IIT has been recently proposed to suppress wild populations of the Mediterranean fruit fly. This method has been successfully tested under laboratory conditions for two major agricultural pests, the Mediterranean fruit fly and the olive fly. It is worthwhile for such an approach to be validated and extended, alone and/or in conjunction with the SIT, to other target insect pest species provided that a genetic sexing strain is established. In artificially infected mosquitoes and Drosophila, Wolbachia infections have been found to affect adult life span, reduce resistance to abiotic conditions, supress responses of the immune system, and interfere with pathogen transmission. These studies led to the application of Wolbachia to reduce fitness of mosquito populations. Similar approaches could be exploited for Tephritid pests. In Tephritid pests without available sexing strains, Wolbachia could potentially be used in IIT approach if female fitness in the field is suppressed, while it is conditionally not expressed in the mass-rearing.

C2. Genus level background Anastrepha: There is limited knowledge regarding tools for the application of IIT and for exploiting fitness effects of reproductive symbionts to control pest populations. Bactrocera: There are no tools for the application of IIT for most species or for exploiting fitness effects of reproductive symbionts to control pest populations. An artificially Wolbachia infected olive fly line can be used for implementing IIT approach provided that a cost-effective rearing is developed. Ceratitis: In C. capitata there are potential tools for the application of the IIT approach. There are artificially Wolbachia infected C. capitata lines including a genetic sexing line that can be used as tools for implementing IIT approach. There are recent data on effects of Wolbachia on medfly demographic and behavioural traits including the Vienna 8 GSS. Also, the performance of the trans-infected Vienna 8 GSS under mass rearing conditions has been assessed and the mating competitiveness of males against wild flies has been determined in semi-field conditions. Dacus: There are no tools for the application of IIT or for exploiting fitness effects of reproductive symbionts to control pest populations.

Rhagoletis: Although IIT has been tested in the past using R. cerasi populations that are naturally infected with different Wolbachia strains, there is no mass rearing system and no genetic sexing lines available. The Wolbachia infection status (including multiple infections) has been determined for many European populations of R. cerasi. There are no tools for the application of IIT or for exploiting fitness effects of reproductive symbionts to control pest populations.

Gaps identified:

C1. General:  There are no genetic sexing strains for most Tephritid pests.  Lack of mass rearing system for Wolbachia infected genetic sexing lines except medfly.  Lack of understanding of the effects of Wolbachia on life history and behavioural traits (with the exception of medfly), as well on the field performance of the released, infected males.  Lack of understanding of effects of irradiation on Wolbachia.  Lack of efficient technology to transfer reproductive symbionts from a donor host to a target pest that can be mass reared.

C2. Genus level background Anastrepha:  Lack of tools for the application of IIT. Bactrocera:  Lack of characterization of the B. oleae Wolbachia infected line under mass rearing conditions.  Lack of semi-field and field assessment of the performance of the B. oleae Wolbachia infected line.  Lack of knowledge regarding fitness effects of Wolbachia in the artificially infected B. oleae line.  There are no IIT tools for other Bactrocera species than B. oleae. Ceratitis:  Lack of open field assessment of the performance of artificially infected (with Wolbachia) C. capitata lines.  Lack of complete understanding of the fitness effects of Wolbachia in artificially infected lines.  Lack of tools for exploiting fitness effects of reproductive symbionts other than Wolbachia to control field populations. Dacus:  Lack of tools for the application of IIT Rhagoletis:  Lack of mass rearing system and genetic sexing lines for R. cerasi.  There are no IIT tools for Rhagoletis species other than R. cerasi.

Individual plans

3.1. Detection, molecular and phenotypic characterization

Genus: Anastrepha Participants: Mariana Mateos, Humberto Martinez-Montoya (USA) Collaborators: Emilio Hernandez, Jorge Toledo (Mexico)

5 years plan  Obtain specimens from four species of Anastrepha (A. ludens, A. striata, A. serpentina, and A. obliqua) from different regions of Mexico. These will be provided by our collaborators at ECOSUR-Tapachula, Chiapas, Mexico.  PCR screening in wild populations of A. ludens, A. obliqua, A. serpentina and A. striata from different geographical origins in Mexico to determine presence/absence of heritable endosymbionts (including Wolbachia and Spiroplasma).  Phenotypic characterization of different Wolbachia strains comparing infected and non-infected populations that share the same genetic background.  Characterization of Wolbachia strains and other heritable endosymbionts.  Evaluation of Wolbachia as potential incompatibility agent in the Mexican fruit fly (A. ludens) and A. obliqua.

Achievements  Anastrepha ludens, A. striata, A. obliqua and A. serpentina specimens were collected in Chiapas, Mexico during the period 2012-2014.  PCR screening for heritable endosymbionts (including Wolbachia and Spiroplasma) was performed in all collected individuals. Wolbachia was found in A. striata, A. obliqua and A. serpentina. Screening results suggest that A. ludens is not naturally infected with Wolbachia; however, sampling was restricted to several specific areas in south Chiapas.  The Wolbachia strain wAstriB was characterized in A. striata with the MLST approach.  Several attempts to transfer Wolbachia from A. striata to A. ludens were performed without success.

Participants: Diego Segura (Argentina) Collaborators: Claudia Conte, Silvia Lanzavecchia (Argentina), Kostas Bourtzis and Caralos Caceres (FAO/IAEA)

5 years plan  Screening of wild populations and laboratory strains from different morphotypes of A. fraterculus to determine presence/absence of Wolbachia strains.  Genetic characterization of the Wolbachia strains found.  Establishment of Wolbachia free A. fraterculus strains

Achievements  Specimens from 3 wild populations from Argentina and one laboratory strain were analysed with wsp gene and 16S RNA gene for Wolbachia detection. All individuals screened were found to be infected. Wolbachia strains were characterized with MLST, wsp-HVR and gltA, groEL and dnaA genes. Two Wolbachia strains (wAfraCast1 and wAfraCast2) were detected with wsp-HVR. No co-infection was detected.

 Four A. fraterculus morphotypes (Brasilian 1, Peruvian, Colombian, Mexican) from 5 laboratory strains have been screened for Wolbachia. All the insects analised so far were infected. Three different srtains of Wolbachia were detected (this activity was carried out in collaboration with Kostas Bourzis, Carlos Cáceres and Antonios Augustinos, IPCL, FAO/IAEA).  Infection of A. fraterculus from Argentina by other reproductive symbionts was carried out. No infection by Spiroplasma sp., Cardinium sp., Ricketsia sp., Arsenophonus sp. or Hamiltonella sp. was detected.

Gaps  More sensitive methods to detect low titer of Wolbachia should be developed.

Recommendations  Assessment of the interaction of reproductive symbionts and gut bacteria and develop protocols that allow avoiding confounding effects of antibiotics/curing procedures.

Participant: Fernando L. Consoli (Brazil) 5 years plan  Determine Wolbachia presence in populations of A. fraterculus from Rio Grande do Sul

Achievements  16S rRNA sequences of Wolbachia have been detected in populations of A. fraterculus from Rio Grande do Sul associated with different host fruits  Analyses of 1400 bp of 16S rRNA indicated this Wolbachia strain shares less than 97% identity with the closest match Wolbachia sequence

Genus: Bactrocera Participants: G. Tsiamis (Greece), Mahfuza Khan (Bangladesh), Ramesh Hire (India) Collaborator : Kostas Bourtzis (FAO/IAEA)

5 years plan  Detect the prevalence of Wolbachia, Cardinium, Rickettsia, Spiroplasma and Arsenophonus strains in populations of the Bactrocera dorsalis complex (B. dorsalis sensu stricto, B. carambolae, B. papayae, and B. philipinnensis) and B. zonata, B. tau, B. cucurbitae, B. correcta, B. scutellaris B. nigrofemoralis, and B. minax.  Genotype the Wolbachia strains  Characterize putative horizontal gene transfer events in B. cucurbitae

Achievements  Detection of Wolbachia, Spiroplasma/Mycoplasma and Cardinium strains in natural populations have been confirmed in speciments of all the above Bactrocera species.  MLST characterization of the Wolbachia strains has been completed.  Prevalence of Wolbachia, Spiroplasma, Arsenophonus, Cardinium, Rickettsia from natural populations from India and Bangladesh.  Genotyping of Wolbachia strains.

Gaps

 in situ hybridisation data

Recommendations  Examine Bactrocera populations from other countries.  Examine presence of fungi, yeast and viruses

Participants: Ramesh Hire, Ashok Hadapad (India) Collaborators: K. Bourtzis (FAO/IAEA), G. Tsiamis (Greece)

5 years plan  Collect B. cucurbitae adults from different agro-climatic conditions and regions for screening Wolbachia presence.  Screening of B. cucurbitae cultures collected from different agro-climatic conditions for Wolbachia infection using MLST approach (wsp, ftsZ) and 16S rDNA specific primers.  Genotype the Wolbachia strains found.

Achievements  Zeugodacus cucurbitae samples were collected from 28 locations of ten Indian states of India during different seasons and climatic condtions.  Eight different Bactrocera species were collected and identified.  The Wolbachia prevalence in B. cucurbitae and other Bactrocera species were studied.  Wolbachia prevalance ranged from 15-62% in Indian Bactrocera species collected during the CRP period.  Genotype the Wolbachia was studied using MLST. Majority of Wolbachia belong to Supergroup A.

Genus: Ceratitis

Participant: Fernando L. Consoli (Brazil) Collaborators: Kostas Bourtzis (FAO/IAEA), Jair Virginio, Julio Walder (Brazil)

5 years plan  Sample populations of C. capitata in several states of Brazil, particularly those representing the major fruit production areas in Brazil  Detect the presence of Wolbachia in each population by diagnostic PCR using the 16S rRNA and wsp gene as targets, testing anywhere from 10 to 20 specimens/population  Characterize Wolbachia in wild C. capitata infected populations by MLST analysis  Assess the molecular diversity of each C. capitata population sampled by identification of the barcode region of the COI  Molecular diversity of W+ and W- C. capitata will be compared at the population (inter population) and individual level (intra population)  W+ C. capitata populations will be selected based on previous data and frequency of Wolbachia infection will be determined

 Compatibility crossing assays between W+ and W- individuals will be carried out within a single population or among populations depending on the results obtained

Achievements  Natural populations of C. capitata were obtained from eight localities representing five different states in Brazil: i) Canavieiras (7 specimens), Jequié (20 specimens), Vitória da Conquista (20 specimens) and Wenceslau Guimarães (7 specimens), all from state of Bahia; ii) Campina Grande (20 specimens), state of Paraíba; iii) Petrolina (18 specimens), state of Pernambuco; iv) Pelotas (20 specimens), state of Rio Grande do Sul; v) Campinas (20 specimens), state of São Paulo;  Samples have been subjected to DNA extraction and diagnostic PCR using the several primers sets for Wolbachia detection (wsp81F/wsp691r, wsp106F/406R, wspecF/wspecR, MLST alleles);  No Wolbachia infection has been detected to be associated with all natural populations examined.

Participant: Aydin Tuncbilek (Turkey) Collaborators: Kostas Bourtzis (FAO/IAEA) and George Tsiamis (Greece)

5 years plan  Collect wild Ceratitis capitata populations from different region of Turkey and over different seasons  Detect presence of Wolbachia strain and other reproductive symbionts

Achievements  Wolbachia, Cardinium and Arsenophonus have not been detect with 16S rDNA gene in all medfly populations tested.  We obtained unexpected bands for Spiroplasma (63F-TK55) in medfly populations.

Gaps  There is need to continue detecting the presence of Spiroplasma and other reproductive symbionts in wild medfly populations.

Recommendations  There is need for a new primer to detect Spiroplasma

Genus Rhagoletis Participant: Nikos Papadopoulos (Greece) Christos Gerofotis, Charalampos Ioannou Collaborators: Kostas Bourtzis (FAO/IAEA), George Tsiamis (Greece)

5 years plan  Determine the demographic and behavioural profile of Wolbachia infected Greek R. cerasi populations  Define the genetic structure of Greek populations of R. cerasi  Genotype the Wolbachia strains in Greek populations of R. cerasi.

 Determine Wolbachia infection status in larvae, pupae and different ages of adult R. cerasi.

Achievements  The demographic profile of three German populations that are differentially infected by multiple Wolbachia strains has been determined.  The demographic profile of Greek populations has been defined.  The genetic structure of Greek populations of R. cerasi has been defined  The Wolbachia strains in Greek populations of R. cerasi has been genotyped.

Gaps  Quantitative and qualitative properties of the Wolbachia infection in different developmental stages of Rhagoletis cerasi and different populations have not been determined yet  The prevelance of other than Wolbachia, reproductive symbions such as Cardinium, Arsenophonus and Spiroplasma in Greek, German, and Turkish populations of Rhagoletis cerasi have not been explored yet  The bacterial community of Rhagoletis cerasi gut has not been explored yet.

Recommendations  Explore the reproductive symbions such as Cardinium, Arsenophonus and Spiroplasma in Greek, German and Turkish population of Rhagoletis cerasi  Define Wolbachia infection in different developmental stages of Rhagoletis cerasi and different populations and determine possible effects of environmental stressors such as temperature

Summary of the achievement at the end of the CRP Species of the genera Anastrepha (Anastrepha ludens, A. striata, A. obliqua and A. serpentina, A. fraterculus) from North and South America, Bactrocera (B. dorsalis sensu stricto, B. carambolae, B. papayae, and B. philipinnensis, B. zonata, B. tau, B. cucurbitae, B. correcta, B. scutellaris B. nigrofemoralis, and B. minax), from South-eastern Asia, Zeugodacus cucurbitae from India, and the Mediterranean fruit fly (C. capitata) from Turkey, Brazil and Argentina have been screened for reproductive endosymbionts and the recorded symbionts have been characterized. Most of the Anastrepha species have been found to be infected with Wolbachia, at various infection rates. Likewise, most of the Bactrocera species as well as Z. cucurbitae have been also found to be infected with Wolbachia and several with Spiroplasma/Mesoplasma. Bactrocera dorsalis populations (from India and Bangladesh) were found to be infected with Cardinium. Putative horizontal gene transfer events have been identified in B. dorsalis, B. zonata and Z. cucurbitae. No Wolbachia infection has been detected in medfly populations from Argentina, Brazil, and Turkey. A case of Spiroplasma infection has been reported in a Turkish C. capitata population. In addition, the genetic structure of Greek and German populations of R. cerasi and of the associated Wolbachia strains has been characterized.

3.2. Interactions of reproductive symbionts and other microorganisms

Genus: Bactrocera

Participant: George Tsiamis (Greece), Mahfuza Khan (Bangladesh) Collaborator: Kostas Bourtzis (FAO/IAEA),

5 years plan  Characterization of the gut symbiotic bacteria using 16S rRNA amplicon sequencing libraries in populations of the Bactrocera dorsalis complex.  Quantitative analysis of the most dominant bacteria of the above species  Characterization of the irradiation impact on the host microbiome (reproductive and gut symbionts)

Achievements  The gut symbiotic bacteria have been characterised using an 16S rRNA amplicon sequencing approach (see 3.1 above)  Characterization of the irradiation impact on the host microbiome has been completed for Z. cucurbitae

Gaps  Changes in the gut microflora have been observed but it is not clear if they are due to the compromization of the immune system or due to changes in the physicochemical conditions of the gastrointestinal tract

Recommendations  Monitor immune response genes using a qPCR approach.  Measure the oxygen, pH, and redox conditions of the gastrointestinal tract before and after irradiation.  Deploy a meta-transcriptomics approach to compare the transcriptional profile of the host and the microbiome before and after irradiation.

Genus: Ceratitis

Participants: Kostas Bourtzis (FAO/IAEA) Collaborators: Nikos Papadopoulos (Greece) and George Tsiamis (Greece)

5 years plan  Characterize the interactions between Wolbachia and gut bacteria in medfly.

Achievements  The interaction between Wolbachia and gut bacteria have been characterized through molecular and confocal microscopy methods in medflies.

Summary of the achievement at the end of the CRP The effect of irradiation on Wolbachia has been studied on transinfected C. capitata lines. The interaction between Wolbachia and the gut bacteria has been studied in transinfected C. capitata lines using molecular and confocal microscopy methods. It seems that Wolbachia suppresses the bacteria community of the alimentary tract of adult medflies. Additional work is required to explore the interaction between reproductive symbionts and gut bacteria in Bactrocera and Anastrepha. 84

3.3. Applications (CI and host fitness manipulations)

Genus Anastrepha Participants: Diego Segura (Argentina) Collaborators: Claudia Conte, Silvia Lanzavecchia (Argentina), Kostas Bourtzis and Caralos Caceres (FAO/IAEA)

5 years plan  Evaluate the role of Wolbachia on reproduction of A. fraterculus.  Evaluate the role of Wolbachia on the interactions between A. fraterculus larvae and Diachasmimorpha longicaudata.

Achievements  Bi-directional cytoplasmic incompatibility between strains of A. fraterculus infected with wAfraCast1 and wAfraCast2 was assessed. No evidences of CI were detected.  The role of Wolbachia on the interaction between A. fraterculus larvae and Diachasmimorpha longicaudata was assessed by comparing the rate of parasitism between infected and cured strains. The strain of A. fraterculus infected by the Wolbachia variant wAfraCast2 showed a significant lower parasitism rate.

Gaps  Evaluation of uni-directional CI in A. fraterculus infected with the two Wolbachia nucleotide variants found in Argentina.

Recommendations  Evaluation of uni-directional CI in all morphotypes of A. fraterculus, and bi- directional CI between morphotypes of A. fraterculus infected by different strains of Wolbachia.  Assessment of the interaction of reproductive symbionts and gut bacteria and develop protocols that allow avoiding confounding effects of antibiotics/curing procedures.  Assesment of interaction among Wolbachia-fruit fly host-parasitoids.

Genus: Bactrocera Participants: Ramesh Hire, Ashok Hadapad (India) 5 years plan  Determine different doses of radiation on B. cucurbitae that assures female sterility in a possible IIT approach.  Performance of B. cucurbitae sterile males with or without Wolbachia infection.

Achievements  Based on the natural infection of Wolbachia in Z. cucurbitae, Wolbachia infected melon fly population under laboratory conditions has been established.  Similarly, uninfected Wolbachia melon fly culture have also been established.  Wolbachia infected and unifected melon fly cultures are being maintaned over 20 generations.

 Wolbachia infected and unifected melon fly cultures were crossed to study the cytoplasmic incompatibility (CI).

Gaps identified • The crosses of Wolbachia infected and uninfected Z. cucurbitae to study CI could not be completed and needs to continue.

Recommendation • Antibody against Wolbachia specific protein could be developed and can be used for localization of Wolbachia. This will help in tracking the Wolbachia during the crossses to induce CI.

Genus: Ceratitis

Participants: Fernando Consoli (Brazil) Collaborators: Kostas Bourtzis (FAO/IAEA), Jair Virgilio (Brazil)

5 years plan  Perform under laboratory conditions mating compatibility assays between the C. capitata Vienna 8 GSS and the naturally occurring Wolbachia-infected and uninfected populations that will be selected from Brazil.  Perform field cage assays to test the efficiency of C. capitata Vienna 8 GSS to suppress selected populations from Brazil.

Participants: Nikos Papadopoulos (Greece) Christos Gerofotis, Charalampos Ioannou Collaborators: Kostas Bourtzis (FAO/IAEA), Carlos Caceres (FAO/IAEA), George Kyritsis, and George Tsiamis (Greece)

5 years plan  Reproductive behaviour of Wolbachia infected medfly lines.  Determine the progress of sexual maturation of both males and females of the Wolbachia-infected and non-infected cohorts. Study the effect of Wolbachia infection on the mating behaviour of both males and females.  Determine remating rates of females mated with Wolbachia infected males  Assess the mating competitiveness of Wolbachia-infected males against wild males to mate with wild females.  Study the interactions between Wolbachia infection and probiotic diets.  Study the interaction between Wolbachia infection and exposure of males to orange oil.  Explore possible impact of Wolbachia infection in the chemical ecology of medfly.  Determine the population increase parameters of Wolbachia infected medfly lines.  Population model  Quantify the performance of the Wolbachia infected lines under small scale mass rearing.  Determine the quality properties and performance of mass reared (under small scale mass rearing) Wolbachia infected lines

 Determine changes in the demographic and behavioural profile of artificially Wolbachia infected medfly lines over time to analyse the evolution of the symbiotic relationships  Evaluate under semi field conditions an IIT approach using the two available Wolbachia infected lines

Achievements  The effect of Wolbachia infection on sexual maturation and the mating behaviour of both males and females has been determined.  The remating rates of females mated with Wolbachia infected males have been characterized for both Wolbachia infected and non-infected female. The full demographic profile of mated and remated Wolbachia infected females has been determined.  The mating competitiveness of Wolbachia-infected males against wild males to mate to wild females has been determined in small laboratory cages.  Small scale mass rearing of Wolbachia infected males have been achieved with moderate success.  Standard quality control tests regarding performance of mass reared Wolbachia infected medfly males including mating competitiveness tests in field cages have been conducted  Population increase parameters have been estimated and can be used for laboratory colonies management and for estimating the impact of Wolbachia-infected released males on wild medfly populations.  The effects of Orange Oil on the mating competitiveness Wolbachia infected males have been explored.  Additional fitness traits of Wolbachia infected individuals, such as cold hardiness, have been addressed.  Effects of Wolachia infection on immatures development in two host fruit and three different temperatures have been explored.  Effect of Wolbachia infected medfly larvae on survival, immature developmental duration and adult longevity of the endoparasitoid Aganaspis daci has been determined.  The performance of the Wolbachia infected lines under small scale mass rearing has been qualified.  The quality properties and performance of mass reared (under small scale mass rearing) Wolbachia infected lines has been determined  The mating performance of the two available Wolbachia infected lines in the context of an IIT application has been evaluated under semi field conditions.  The population increase parameters of Wolbachia infected medfly lines have been estimated.

Gaps Determine the interactions between Wolbachia infection and probiotic diets in both larvae and adults. Pilot tests of IIT releasing Wolbachia infected males have not been atempted

Recomendations

Determine the interactions between Wolbachia infection and the use of probiotic diets at both larvae and adult medflies.

Summary of the achievement at the end of the CRP Wolbachia infected and uninfected melon fly cultures were crossed and cytoplasmic incompatibility (CI) has been determined. Similar trials have been conducted to check the existence of possible bi-directional CI in A. fraterculus. The effects of different Wolbachia strains on demographic, reproductive and behavioral traits of transinfected medfly lines have been determined in detail, including interactions between two medfly genetic backgrounds and different Wolbachia strains. The population increase parameters of Wolbachia infected medfly lines have been estimated. Small scale mass rearing of Wolbachia infected males have been achieved and mating competitiveness experiments in field cages have been conducted. In addition, the response of Wolbachia infected medfly lines to a range of environmental stresses (cold, host fruit) have been determined.

Table 3. Status of different Fruit flies of economic importance with respect their infection with the endosinbiont Wolbachia spp

Genus Species Location Wolbachia MLST status Strain(s)

Anastrepha ludens Mexico Not detected striata Mexico Detected wAstriB obliqua Mexico Detected * serpentina Mexico Detected * fraterculus Argentina Detected wAfra (Brazilian 1 Cast1, morphotype) wAfra Cast2 Bactrocera cucurbitae India/Bangladesh Detected * zonata India/Bangladesh Detected * tau India/Bangladesh Detected * dorsalis India/Bangladesh Detected * correcta India Detected * tryoni Australia Detected/ low prevalence neohumeralis Australia Detected/ low prevalence scutellaris India Detected nigrofemoralis India Deteted Ceratitis capitata Brazil/Turkey Not detected Rhagoletis cerasi Greece Detected wCer1, wCer2, wCer4, wCer5

Table 4 List of Bacterial species found during current CRP (see Table 2 for previous studies)

Fly species Bacterial Species (culture Life stage Comments Principal dependent) (Beneficial, investigator Pathogenic)

Anastrepha Morganella morganii (22 Larvae and egg pathogenic Schuenzel ludens isolates) et al

(USA)

Providencia alcafaciens (2 Larvae pathogenic " isolates)

Providencia sp. (2 isolates) Larvae pathogenic "

Providencia rettgeri Larvae pathogenic "

Acinetobacter sp. Larvae and neutral " diet

Alcaliginaceae Larvae and neutral " diet

Enterobacter kobei Larvae and neutral " diet

Grimontella senegalensis Larvae and neutral " diet

Kerstersia gyiorum Larvae and neutral " diet

Klebsiella pneumoniae Larvae and neutral " diet

Lactococcus lactis Larvae and neutral " diet

Pseudomonas aeruginosa Larvae and neutral " diet

Pseudomonas monteilii Larvae and neutral " diet

Serratia marcescens Larvae and neutral "

diet

Serratia rubidaea Larvae and neutral " diet

Stenotrophomonas maltophilia Larvae and neutral " diet

Anastrepha Enterobacter cloacae egg beneficial Melgar et ludens al (Black (Guatemala pupae GSS) )

Aeromonas hydrophila egg not " determined

Proteus stuartii egg not " determined

Klebsiella pneumoniae egg beneficial "

Enterobacter agglomerans egg beneficial "

Enterobacter aerogenes larvae beneficial "

Providencia stuartii pupae not " determined

Providencia alcalifaciens 1-2 egg not " determined

Anastrepha Raoultella ornithinolitica infested guava not Melgar et ludens larvae determined al (wild) (Guatemala )

Enterobacter agglomerans infested guava beneficial " larvae

Tatumella sp larvae not " determined

Enterobacter aerogenes infested guava beneficial " larvae

Klebsiella oxytoca infested guava beneficial " larvae

Enterobacter cloacae infested guava beneficial " larvae

Anastrepha Enterobacter cloacae Adult (midgut, not Hernández ludens 8 days-old) determined et al (Lab) (Mexico)

Serratia marcescens Adult (midgut, not " 8 days-old) determined

Citrobacter freundii Adult (midgut, not " 8 days-old) determined

Morganella morganii Adult (midgut, not " 8 days-old) determined

Erwinia spp. Adult (midgut, not " 8 days-old) determined

Providencia rettgeri Adult (midgut, not " 8 days-old) determined

Staphylococcus aureus Adult (midgut, not " 8 days-old) determined

Pantoea aglomerans Adult (midgut, not " 8 days-old) determined

Providencia rettgeri Adult (midgut, not " 8 days-old) determined

A. Acinetobacter bereziniae Mature larva not Consoli et fraterculus determined al, (Brazil) (wield)

Arthrobacter soli Mature larva not " determined

Cedecea davisae Mature larva not " determined

Comamonas koreensis Mature larva not " determined

Empedobacter brevis Mature larva not " determined

Enterobacter aerogenes Mature larva not " determined

Lactococcus lactis subsp. lactis Mature larva not " determined

Acinetobacter bereziniae Newly- not " emerged adult determined

Brevundimonas olei Newly- not " emerged adult determined

Cedecea davisae Newly- not " emerged adult determined

Citrobacter braakii Newly- not " emerged adult determined

Comamonas koreensis Newly- not " emerged adult determined

Empedobacter brevis Newly- not " emerged adult determined

Enterobacter asburiae Newly- not " emerged adult determined

Enterobacter hormaechei Newly- not " emerged adult determined

Enterobacter mori Newly- not " emerged adult determined

Enterococcus faecalis Newly- not " emerged adult determined

Klebsiella michiganensis Newly- not " emerged adult determined

Klebsiella oxytoca Newly- not " emerged adult determined

Kluyvera cryocrescens Newly- not " emerged adult determined

Morganella morganii subsp. Newly- not " sibonii emerged adult determined

Ochrobactrum Newly- not " pseudogrignonense emerged adult determined

Pantoea rodasii Newly- not " emerged adult determined

Providencia rettgeri Newly- not " emerged adult determined

Providencia vermicola Newly- not " emerged adult determined

Pseudochrobactrum Newly- not " saccharolyticum emerged adult determined

Raoultella planticola Newly- not " emerged adult determined

Serratia marcescens subsp. Newly- not " sakuensis emerged adult determined

Staphylococcus sciuri subsp. Newly- not " sciuri emerged adult determined

A. Adult Not Segura et fraterculus determined al., (LAB) Enterococcus sp. (Argentina)

Adult not " Enterobacter sp. determined

A. Adult not " fraterculus determined (wild) Klebsiella sp.

Kluyvera sp. Adult not "

determined

Adult not " Enterobacter sp. determined

Adult not " Erwinia sp. determined

Adult not " Citrobacter sp. determined

Anastrepha Enterobacter cloacae Adult (midgut, beneficial Hernández obliqua 8 days-old) et al

(Mexico)

Serratia marcescens Adult (midgut, pathogenic " 8 days-old)

Citrobacter freundii Adult (midgut, neutral " 8 days-old)

Erwinia spp. Adult (midgut, neutral " 8 days-old)

Providencia rettgeri Adult (midgut, beneficial " 8 days-old)

Staphylococcus aureus Adult (midgut, neutral " 8 days-old)

Klebsiella varícola Adult (midgut, not " 8 days-old) determined

Enterobacter asburiae Adult (midgut, not " 8 days-old) determined

Pluralibacter gergoviae Adult (midgut, not " 8 days-old) determined

Klebsiella pneumoniae Adult (midgut, not " 8 days-old) determined

Providencia rettgeri Adult (midgut, beneficial " 8 days-old)

Staphylococcus pasteuri Adult (midgut, not " 8 days-old) determined

Bacillus spp. Adult (midgut, beneficial " 8 days-old)

Anastrepha Serratia marcescens Adult (midgut, pathogenic Hernández serpentina 8 days-old) et al

(Mexico)

Pantoea aglomerans Adult (midgut, beneficial " 8 days-old)

Staphylococcus xylosus Adult (midgut, neutral " 8 days-old)

Providencia rettgeri Adult (midgut, beneficial " 8 days-old)

Staphylococcus aureus Adult (midgut, neutral " 8 days-old)

Chryseobacterium indologenes Adult (midgut, neutral " 8 days-old)

Anastrepha Enterobacter cloacae Adult (midgut, Beneficial Hernández striata 8 days-old) et al. (laboratory) (Mexico)

Serratia marcescens Adult (midgut, Not " 8 days-old) determined

Citrobacter freundii Adult (midgut, Not " 8 days-old) determined

Erwinia spp. Adult (midgut, Not " 8 days-old) determined

Providencia rettgeri Adult (midgut, Not " 8 days-old) determined

Staphylococcus aureus Adult (midgut, Not " 8 days-old) determined

Klebsiella varícola Adult (midgut, Not " 8 days-old) determined

Enterobacter asburiae Adult (midgut, Not " 8 days-old) determined

Pluralibacter gergoviae Adult (midgut, Not " 8 days-old) determined

Klebsiella pneumoniae Adult (midgut, Not " 8 days-old) determined

Providencia rettgeri Adult (midgut, Not " 8 days-old) determined

Staphylococcus pasteuri Adult (midgut, Not " 8 days-old) determined

Klebsiella spp. Adult (midgut, Beneficial " 8 days-old)

Pseudomonas aeruginosa Adult (midgut, Beneficial " 8 days-old)

Enterococcus faecalis Adult (midgut, Neutral " 8 days-old)

Bacillus spp. Adult (midgut, Not " 8 days-old) determined

Bactrocera Acinetobacter baumannii/ Adult stage not Khan et al. cucurbitae calcoceticus (gut bacteria) determined (Banglades h) (10 days old female)

Alcaligenes faecalis Adult stage not " (gut bacteria) determined

Citrobacter youngae (10 days old tested " female)

Citrobacter Roseri/analonaticus Adult stage not " (gut bacteria) determined

Enterobacter cloacae (10 days old not " female) determined

Enterococcus faecium Adult stage not " (gut bacteria) determined

Pantoea spp 4 (10 days old tested " female)

Pseudomonas putida Adult stage not " (gut bacteria) determined

Providencia rettgerri (10 days old not " female) determined

Kluyvera spp Adult stage not " (gut bacteria) determined

Klebsiella oxytoca (10 days old not " female) determined

Delftia acidovorans Larval stage not " (gut bacteria) determined

(third instar

Enterobacter cloacae Larval stage not " (gut bacteria) determined

(third instar

Providencia stuartii Larval stage not " (gut bacteria) determined

(third instar

Pseudomonas putida Larval stage not " (gut bacteria) determined

(third instar

Serratia marcescens Larval stage tested " (gut bacteria)

(third instar

Stenotrophomonas maltophilia Larval stage not " (gut bacteria) determined

(third instar

Klebsiella pneumoniae ssp Larval stage not " pneumoniae (gut bacteria) determined

(third instar

Pantoea spp 3 Larval stage tested " (gut bacteria)

(third instar

Bactrocera Acinetobacter baumannii/ Adult stage not Khan et al. dorsalis calcoaceticus (gut bacteria) determined (Banglades h) (10 days old female)

Acinetobacter junii/johnsonii Adult stage not " (gut bacteria) determined

(10 days old female)

Alcaligenes faecalis Adult stage not " (gut bacteria) determined

(10 days old female)

Citrobacter youngae Adult stage tested " (gut bacteria)

(10 days old female)

Proteus mirabilis Adult stage not " (gut bacteria) determined

(10 days old female)

Pseudomonas putida Adult stage not "

(gut bacteria) determined

(10 days old female)

Klebsiella oxytoca Adult stage not " (gut bacteria) determined

(10 days old female)

Klebsiella pneumoniae ssp Adult stage not " pneumoniae (gut bacteria) determined

(10 days old female)

Enterococcus avium Adult stage not " (gut bacteria) determined

(10 days old female)

Acinetobacter baumannii/ Adult stage not " calcoceticus (gut bacteria) determined

(10 days old female)

Alcaligenes faecalis Adult stage not " (gut bacteria) determined

(10 days old female)

Delftia acidovorans Adult stage not " (gut bacteria) determined

(10 days old female)

Enterobacter cloacae Adult stage not " (gut bacteria) determined

(10 days old

100

female)

Pseudomonas putida Adult stage not " (gut bacteria) determined

(10 days old female)

Proteus mirabilis Adult stage not " (gut bacteria) determined

(10 days old female)

Staphylococcus haemolyticus Adult stage tested " (gut bacteria)

(10 days old female)

Stenotrophomonas maltophilia Adult stage not " (gut bacteria) determined

(10 days old female)

Ceratitis Enterobacter cloacae infested guava beneficial Melgar et capitata al. (wild) (Guatemala )

Enterobacter agglomerans infested guava beneficial "

Klebsiella oxytoca infested guava beneficial "

Serratia marscescens infested guava pathogenic "

Morganella morganii infested guava pathogenic "

Providencia rettgeri infested guava pathogenic "

Ceratitis Enterobacter aerogenes mature larva, beneficial Melgar et capitata egg al. Viena 8 (Guatemala D53-

101

Toliman ) (Lab)

Enterobacter cloacae larva, egg beneficial "

Klebsiella oxytoca larva, egg beneficial "

Pseusomonas larva, egg beneficial " flourescens/putida

Serratia marcescens larva, egg, pathogenic " adult insect adult

Morganella morganii larva, egg pathogenic " larvae

Proteus stuartii larva not " determined

Chromobacterium violaceum egg pathogenic "

Enterobacter gergoviae larva not " determined

Providencia alcalifaciens 1-2 egg not " determined

Enterobacter agglomerans larva, egg beneficial "

Klebsiella rhinoscleromatis egg not " determined

Klebsiella pneumoniae egg beneficial "

Klebsiella rhinoscleromatis larva, egg not " determined

Ceratitis Klebsiella oxycota, Adult (gut API Tuncbilek capitate bacteria, 10 et al. (wild) days old) (Turkey)

K. pneumoniae Adult (gut API " bacteria, 10 days old)

Enterobacter cloacae Adult (gut API "

102

bacteria, 10 days old)

E. cancerogenus Adult (gut API " bacteria, 10 days old)

Enterococcus faecalis Adult (gut Single " bacteria, 10 colony days old) PCR

Klebsiella oxycota Adult (gut not " bacteria, 10 determined days old)

Morganella morganii Adult (gut not " bacteria, 10 determined days old)

Ceratitis Bacillus sp. Mature larvae not Yáñez et al. capitata determined (Chile) Vienna 8 (Lab.)

Morganella sp. Mature larvae not " determined

Providencia sp. Mature larvae not " determined

Serratia sp. Mature larvae not " determined

Enterobacter sp. Mature larvae not " determined

Citrobacter sp. Mature larvae not " determined

Klebsiella sp. Mature larvae not " determined

Serratia sp. Pupae not "

103

determined

Enterobacter sp. Pupae not " determined

Citrobacter sp. Pupae not " determined

Klebsiella sp. Pupae not " determined

Citrobacter sp. Irradiated not " pupae determined

Providencia sp. Irradiated not " pupae determined

Serratia sp. Irradiated not " pupae determined

Klebsiella sp. Irradiated not " pupae determined

Stenotrophomonas sp. Irradiated not " pupae determined

Serratia sp. Adults not " determined

Pseudomonas sp. Adults not " determined

Klebsiella sp. Adults not " determined

Enterobacter sp. Adults not " determined

Citrobacter sp. Adults not " determined

Stenotrophomonas sp. Irradiated not " adults determined

Klebsiella sp. Irradiated not "

104

adults determined

Citrobacter sp. Irradiated not " adults determined

Serratia sp. Irradiated not " adults determined

Pseudomonas sp. Irradiated not " adults determined

Providencia sp. Irradiated not " adults determined

Enterobacter sp. Irradiated not " adults determined

Ceratitis Citrobacter sp. larvae not Yáñez et al. capitata determined (Chile) Vienna 8 (- invD53/Toli man 99) (Lab.)

Enterobacter sp. larvae not " determined

Serratia sp. larvae not " determined

Klebsiella sp. larvae not " determined

Providencia sp. Pupae not " determined

Stenotrophomonas sp. Pupae not " determined

Klebsiella sp. Irradiated not " pupae determined

Citrobacter sp. Irradiated not " pupae determined

105

Serratia sp. Irradiated not " pupae determined

Providencia sp. Irradiated not " pupae determined

Stenotrophomonas sp. Irradiated not " pupae determined

Citrobacter sp. Adults not " determined

Serratia sp. Adults not " determined

Klebsiella sp. Adults not " determined

Enterobacter sp. Adults not " determined

Pseudomonas sp. Adults not " determined

Enterobacter sp. Irradiated not " adults determined

Klebsiella sp. Irradiated not " adults determined

Stenotrophomonas sp. Irradiated not " adults determined

Citrobacter sp. Irradiated not " adults determined

Providencia sp. Irradiated not " adults determined

Pseudomonas sp. Irradiated not " adults determined

Serratia sp. Irradiated not " adults determined

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Ceratitis Bacillus sp. Adults not " capitata determined wild (Argentina)

Klebsiella sp. Adults not " determined

Kluyvera sp. Adults not " determined

Ceratitis Klebsiella oxytoca infested peach not Gª de capitata determined Oteyza et (wild) al. (Spain)

Enterobacter cloacae infested peach not " determined

E. gergoviae infested peach not “ determined

Pantoea agglomerans infested peach not " determined

Providencia rettgeri infested peach not " determined

Buttiauxella noackiae infested peach not " determined

Serratia marcescens infested peach not " determined

Klebsiella oxytoca infested not " orange determined

Enterobacter agglomerans infested not " orange determined

Pantoea agglomerans infested not " orange determined

Providencia rettgeri infested not " orange determined

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Klebsiella oxytoca larvae not " determined

Enterobacter cloacae larvae not " determined

Morganella morganii larvae not " determined

Klebsiella oxytoca adult not " determined

Enterobacter cloacae adult not " determined

Morganella morganii adult not " determined

Ceratitis Enterobacter cloacae egg not Gª de capitata V8 determined Oteyza et (mass al. (Spain) reared)

E. aerogenes egg not “ determined

Pseudomonas putida egg not " determined

Serratia marcescens egg not " determined

Staphylococcus saprophyticus egg not " determined

Providencia rettgeri larvae not " determined

Morganella morganii larvae not " determined

Pseudomonas putida larvae not " determined

Klebisella oxytoca larvae not "

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determined

Klebisella pneumoniae larvae not " determined

Raoultella ornithinolytica larvae not " determined

Pantoea agglomerans larvae not " determined

Enterobacter cloacae larvae not " determined

E. gergoviae larvae not “ determined

Staphylococcus saprophyticus larvae not “ determined

Citrobacter freundii larvae not " determined

Pluralibacter adult Not “ determined

Klebsiella oxytoca adult not " determined

K. pneumonia adult not “ determined

Enterobacter cloacae adult not " determined

Providencia rettgeri adult not " determined

Providencia stuartii adult not “ determined

Morganella morganii adult not " determined

Citrobacter freundii adult not "

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determined

Pantoea agglomerans adult not " determined

Kluyvera cryocrescens adult not " determined

Buttiauxella noackiae adult not " determined

A. Bacterial Species (culture Life stage Comments Principal fraterculus independent) – 16S rRNA (Beneficial, investigator (Morphoty sequencing Pathogenic) pe I, wild)

Acetobacter fabarum Late larva not Consoli et determined al. (Brazil)

Acetobacter indonesiensis Late larva not " determined

Acetobacter nitrogenifigens Late larva not " determined

Achromobacter xylosoxidans Late larva not " determined

Acinetobacter berezinae Late larva not " determined

Alcaligenes faecalis subsp. Late larva not " faecalis determined

Aquabacterium commune Late larva not " determined

Aquicella siphonis Late larva not " determined

Bradyrhizobium liaoningense Late larva not " determined

Bradyrhizobium oligotrophicum Late larva not " determined

110

Bradyrhizobium pachyrhizi Late larva not " determined

Bradyrhizobium rifense Late larva not " determined

Caulobacter segnis Late larva not " determined

Chryseobacterium Late larva not " yeoncheonense determined

Chthoniobacter flavus Late larva not " determined

Comamonas koreensis Late larva not " determined

Deinococcus apachensis Late larva not " determined

Denitratisoma oestradiolicum Late larva not " determined

Elusimicrobium minutum Late larva not " determined

Enterobacter asburiae Late larva not " determined

Enterobacter hormaechei Late larva not " determined

Enterobacter oryzae Late larva not " determined

Escherichia coli Late larva not " determined

Glaciimonas singularis Late larva not " determined

Gloeobacter violaceus Late larva not " determined

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Gluconobacter cerinus Late larva not " determined

Gluconobacter kondonii Late larva not " determined

Herbaspirillum Late larva not " chlorophenolicum determined

Hydrogenophilus hirschii Late larva not " determined

Leclercia adecarboxylata Late larva not " determined

Leuconostoc Late larva not " pseudomesenteroides determined

Massilia suwonensis Late larva not " determined

Methylobacterium aquaticum Late larva not " determined

Methylobacterium Late larva not " organophilum determined

Methylobacterium podarium Late larva not " determined

Microbacterium ginsengisoli Late larva not " determined

Novosphingobium Late larva not " aromaticivorans determined

Owenweeksia hongkongensis Late larva not " determined

Pedomicrobium australicum Late larva not " determined

Phycisphaera mikurensi Late larva not " determined

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Polyangium brachysporum Late larva not " determined

Propionibacterium acnes Late larva not " determined

Pscinibacter aquaticus Late larva not " determined

Pseudochrobactrum lubricantis Late larva not " determined

Pseudomonas geniculata Late larva not " determined

Raoultella planticola Late larva not " determined

Rhizomicrobium electricum Late larva not " determined

Schlegelella aquatica Late larva not " determined

Sediminibacterium salmoneum Late larva not " determined

Serratia marcescens subsp. Late larva not " sakuensis determined

Sphingomonas echinoides Late larva not " determined

Sphingomonas leidyi Late larva not " determined

Steroidobacter denitrificans Late larva not " determined

Tatumella ptyseos Late larva not " determined

Thioalkalimicrobium aerophilum Late larva not " determined

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Wolbachia pipientis Lat Newly- not " emerged adult determined e larva

Acinetobacter berezinae Newly- not Consoli et emerged adult determined al. (Brazil)

Alcaligenes faecalis subsp. Newly- not " faecalis emerged adult determined

Arthrobacter mysorens Newly- not " emerged adult determined

Brevundimonas olei Newly- not " emerged adult determined

Cedecea davisae Newly- not " emerged adult determined

Cedecea neteri Newly- not " emerged adult determined

Chryseobacterium Newly- not " arthrosphaerae emerged adult determined

Comamonas testosteroni Newly- not " emerged adult determined

Commensalibacter intestini Newly- not " emerged adult determined

Enterobacter asburiae Newly- not " emerged adult determined

Enterococcus casseliflavus Newly- not " emerged adult determined

Enterococcus faecalis Newly- not " emerged adult determined

Escherichia fergusonii Newly- not " emerged adult determined

Hydrogenophilus hirschii Newly- not " emerged adult determined

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Klebsiella michiganensis Newly- not " emerged adult determined

Ochrobactrum haematophilum Newly- not " emerged adult determined

Ochrobactrum Newly- not " pseudogrignonense emerged adult determined

Ochrobactrum tritici Newly- not " emerged adult determined

Providencia vermicola Newly- not " emerged adult determined

Pseudochrobactrum lubricantis Newly- not " emerged adult determined

Pseudomonas alcaliphila Newly- not " emerged adult determined

Pseudomonas beteli Newly- not " emerged adult determined

Raoutella ornithinolytica Newly- not " emerged adult determined

Rhizobium leguminosarum Newly- not " emerged adult determined

Rhodobacter veldkampii Newly- not " emerged adult determined

Serratia marcescens subsp. Newly- not " sakuensis emerged adult determined

Sphingomonas alpina Newly- not " emerged adult determined

Staphylococcus sciuri subsp. Newly- not " sciuri emerged adult determined

Wolbachia pipientis Newly- not " emerged adult determined

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Yokenella regensburgei Newly- not " emerged adult determined

A. Enterobacter sp. Adult Not Segura et fraterculus determined al., (LAB) (Argentina) Serratia sp. Adult Not " determined Stenotrophomonas sp. Adult Not " determined A. Klebsiella sp. Adult Not " fraterculus determined (wild) Serratia sp. Adult Not " determined Kluyvera sp. Adult Not " determined Enterobacter sp. Adult Not " determined Citrobacter sp. Adult Not " determined Stenotrophomonas sp. Adult Not " determined B. tryoni Asaia spp. 2nd – 3rd instars Beneficial Reynolds & larvae larvae Deutscher (wild) (Australia) Acetobacter spp. 2nd – 3rd instars Not " larvae determined Acetobacter orientalis 2nd – 3rd instars Not " larvae determined Enterobacter spp. 2nd – 3rd instars Not " larvae determined Enterobacter cloacae complex 2nd – 3rd instars Not " larvae determined Tatumella sp. 2nd – 3rd instars Not " larvae determined Tatumella/Kluyvera/Enterobact 2nd – 3rd instars Not " er larvae determined Gluconobacter spp. 2nd – 3rd instars Not " larvae determined Janibacter sp. 2nd – 3rd instars Not " larvae determined Methylobacterium spp. 2nd – 3rd instars Not " larvae determined Pantoea/Erwinia spp. 2nd – 3rd instars Not " larvae determined

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Providencia sp. 2nd – 3rd instars Not " larvae determined Tatumella sp. 2nd – 3rd instars Not " larvae determined B. tryoni Asaia spp. 2nd – 3rd instars Not Reynolds & (domesticat larvae determined Deutscher ed) (Australia) Janibacter 2nd – 3rd instars Not " larvae determined Methylobacterium 2nd – 3rd instars Not " larvae determined Propionibacterium acnés spp. 2nd – 3rd instars Not " larvae determined Pseudomonas sp. 2nd – 3rd instars Not " larvae determined Xanthomonas citri/axonopodis 2nd – 3rd instars Not " larvae determined B. Tryoni Asaia sp. Adult Not Reynolds & determined Woruba (Australia) Enterobacter sp. Adult Not " determined Morganella sp. Adult Not " determined Proteus sp. Adult Not " determined Providencia sp. Adult Not " determined Serratia sp. Adult Not " determined B. tryoni Enterobacter sp. Teneral adult Not Reynolds & adults ( determined Woruba University (Australia) of Western Sydney laboratory colony) Morganella sp. Teneral adult Not " determined Proteus sp. Teneral adult Not " determined Providencia sp. Teneral adult Not " determined Serratia sp. Teneral adult Not " determined B. tryoni Asaia sp. Teneral adult Not Reynolds &

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adults determined Woruba (Fruit Fly (Australia) Production Facility) Ceratitis Klebsiella sp. Larvae Not Gª de capitata determined Oteyza et (wild) al. (Spain) Leclercia sp. Larvae Not “ determined Enterobacter sp. Larvae Not “ determined Gluconobacter sp. Larvae Not “ determined Serratia sp. Larvae Not “ determined Acetobacter sp. Larvae Not “ determined Erwinia sp. Larvae Not “ determined Raoultella sp. Larvae Not “ determined Bifidobacterium sp. Larvae Not “ determined Tatumella sp. Larvae Not “ determined Cronobacter sp. Adult Not “ determined Enterobacter sp. Adult Not “ determined Providencia sp. Adult Not “ determined Leclercia sp. Adult Not “ determined Raoultella sp. Adult Not “ determined Ceratitis Providencia sp. larvae Not Gª de capitata determined Oteyza et (V8 mass al. (Spain) reared) Moellerella sp. larvae Not “ determined Morganella sp. larvae Not “ determined Klebsiella sp. larvae Not “ determined

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Leclercia sp. larvae Not “ determined Providencia sp. Sterile adult Not “ determined Lactococcus sp. Sterile adult Not “ determined Klebsiella sp. Sterile adult Not “ determined Enterobacter sp. Sterile adult Not “ determined Raoultella sp. Sterile adult Not “ determined Cronobacter sp. Sterile adult Not “ determined Kluyvera sp. Sterile adult Not “ determined Citrobacter sp. Sterile adult Not “ determined Buttiauxella sp. Sterile adult Not “ determined Providencia sp. Adult Not “ determined Lactococcus sp. Adult Not “ determined Morganella sp. Adult Not “ determined Klebsiella sp. Adult Not “ determined Enterobacter sp. Adult Not “ determined Cronobacter sp. Adult Not “ determined Enterococcus sp. Adult Not “ determined Buttiauxella sp. Adult Not “ determined Citrobacter sp. Adult Not “ determined

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LOGICAL FRAMEWORK

Project Design Elements Verifiable Indicators Means of Verification Important Assumptions Overall Objective: N/A N/A Fruit and vegetable production in The objective of the project is to Member States continue to suffer understand and harness the role major losses to endemic and that microorganisms play in the introduced pests. larval and adult biology of International trade in fruit and selected key fruit and vegetable vegetable commodities will pests to improve the SIT and continue to increase and be related emerging techniques. For disrupted by pests requiring example as part of area-wide expensive post-harvest and integrated, pest outbreak situations quarantine measures. and prophylactic pest management The increasing demand for area- programmes applied against such wide integrated pest management pests. approaches to control fruit and vegetable pests, including where appropriate the SIT and related emerging techniques as non- polluting suppression/eradication components, mandates improvement of the cost- effectiveness of this environment- friendly sustainable approach.

Specific Objectives: N/A N/A 1. To develop methods of using 1. Microorganisms contribute to beneficial bacteria to replace the nutrition and health of their costly ingredients in larval and insect hosts and can be used to

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adult diets in insect mass rearing reduce the costs and increase facilities And improve the quality efficiency of mass-rearing. of the mass reared insects 2. Radiation affects symbiotic 2. To determine the effect of associations. radiation on the symbiotic associations in target species. 3. Probiotic supplements to the 3. To explore the use of symbionts pre-release adult diet of sterile as probiotics provided to adult males can improve their sterile males before their release to subsequent sexual performance. significantly improve sterile male performance. 4. Symbiotic associations can be 4. To harness symbiotic harnessed to manipulate host associations towards the reproduction and reduce target reproductive manipulation and populations. suppression of target populations.

Outcomes: 1. Costs of mass-rearing of target 1. A protocol developed and 1. Mass rearing data and cost- 1. Facilities are eager to reduce fruit pests reduced by validated under mass-rearing benefit analysis. mass-rearing costs. The protocol incorporating microorganisms in conditions. developed is effective and can larval and adult diets. help reduce these costs.

2. A better understanding of the 2. Development of a monitoring 2. Data collected upon the 2. Irradiation detrimentally affects impact of radiation on symbiotic protocol for the characterization of implementation of the monitoring the mass-reared insects’ symbiotic associations of mass-reared post-radiation symbiotic protocol. associations. There is a need to insects. communities. understand and minimize these effects.

3. Performance of sterile males is

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3. Improved sexual performance 3. A protocol developed and 3. Data collected and cost-benefit not optimal and can be improved. of sterile males based upon validated under operational analysis completed. Symbiont-based probiotic probiotic supplements to pre- conditions. supplements to pre-release adult release adult diet. diet can improve sterile male sexual performance.

4. Symbiont-based population 4. An additional control tool that 4. An additional control tool 4. Data collected and feasibility control strategies can be is based on symbiotic associations designed and validated. analysis completed. developed and these novel control and complementary to the SIT is tools will complement the SIT. available.

Outputs: 1.a. Egg and larval microflora of 1.a. Characterized in at least 4 1.a. Reports and peer reviewed 1.a. The microbial assemblages of pests (natural and mass-reared) target species. publications. mass-reared insects are species characterized. specific and amenable to characterization by classical and molecular microbiological approaches.

1.b. The ability of beneficial 1.b. Explored in the mass-reared 1.b. Reports and peer reviewed 1.b. Beneficial bacteria can be bacteria to reduce production costs larval diets of at least two target publications. cultured and harnessed to replace of mass-reared larval and adult species. costly ingredients in larval and diets is explored. adult diets, such that high quality mass-reared insects are produced. Gel-based and liquid diets will allow the elimination of bulking agents that introduce and perpetuate deleterious microorganisms in mass-rearing facilities. The beneficial bacteria can also serve as barriers against

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deleterious microorganisms.

2. The effect of radiation on the 2. Determined in at least four 2. Reports and peer reviewed 2. Irradiation damages symbiotic symbiotic associations is target mass-reared species. publications. associations in a dose-dependent determined. manner.

3.a. Adult associated microflora of 3.a. Characterized in at least four 3.a. Reports and peer reviewed 3.a. The microbial assemblages of pests (natural and mass-reared) target species. publications. adult insects are species-specific characterized. and amenable to characterization by classical and molecular microbiological approaches.

3.b The impact of symbiont-based 3.b Determined for at least three 3.b. Reports and peer reviewed 3.b. Symbionts can be used as probiotic supplements to adult target species. publications. probiotic supplements in diets pre-release diet on sterile male provided to sterile males before sexual performance is determined. their release to improve performance.

4.a. Microorganisms that affect 4.a. Characterized in at least two 4.a. Reports and peer reviewed 4.a. Microorganisms that are host reproduction are target species. publications. potential reproductive characterized. manipulators are amenable to characterization by molecular microbiological approaches.

4.b. Characterized in at least one 4.b. Reports and peer reviewed 4.b. The tripartite interactions of 4.b. The tripartite interactions of target species. publications. host-parasitoid-symbionts affect host-parasitoid-symbionts the host fitness and are amenable characterized. to manipulation.

4.c. Explored in at least one target 4.c. Reports and peer reviewed 4.c. Symbiont-based population 4.c. Population suppression by species. publications. control strategies can be

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microorganisms that manipulate developed against target pest host reproduction is explored. species.

Activities: 1. Hold Consultants Meeting. 1. Consultants meeting held 1. Report of Consultants Meeting 1. CRP proposal approved by February 2011. and CRP proposal prepared. IAEA committee.

2. Announce project amongst 2. CRP announced, and research 2. Issued contracts and 2. Proposals submitted and established entomologists and contracts and agreements agreements. approved by IAEA committee. symbiologists researchers and fruit submitted, evaluated and and vegetable pest control forwarded to IAEA committee. programmes and establish CRP.

3. Organize first RCM to plan, 3. 1st RCM held in mid-2012. 3. Working material printed and 3. Research activities commence. coordinate and review research distributed for 1st RCM. Reports published and distributed activities (2nd quarter 2012). following each RCM.

4. Carry out R&D and draft 4. Research carried out by contract 4. Reports and publications. 4. Renewal requests and continued technical protocols. and agreement holders. funding of RCM’s and CRP.

5. Second RCM to present data 5. 2nd RCM held in May 2014, 5. Working material printed and 5. Research activities continue; and coordinate future research as Bangkok, Thailand. distributed for 2nd RCM; progress satisfactory. required (early 2014). Research published in scientific literature and disseminated to member states and scientific community.

6. In conjunction with second 6. Workshop held in May, 2014. 6. Workshop report. 6. There is need for training; RCM, hold workshop on "Novel Harmonized procedures and techniques and instructors are tools for the characterization and trainees capable of implementing available.

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bioinformatics analysis of insect novel techniques. symbiotic communities". 7. Research carried out by contract 7. Continue R&D. and agreement holders. 7. Reports and publications. 7. Renewal requests and continued funding of RCM’s and CRP. 8. Mid-CRP review carried out. 8. Review the CRP after its third 8. Report of mid-CRP review. 8. Mid-CRP review by Agency year. committee is positive. 9. 3rd RCM held in 2015. 9. Third RCM to present data and 9. Working material printed and 9. Mid-CRP review approved by coordinate future research as distributed for 3rd RCM; Research IAEA committee. Research required (late 2015). published in scientific literature activities continue; progress and disseminated to member states satisfactory. and scientific community. 10. Workshop held 2015. 10. In conjunction with third Harmonized procedures and 10. Workshop report. 10. There is need for training; RCM, hold workshop on trainees with novel techniques. techniques, equipment and "Incorporating bacteria to larval instructors are available. and pre-release adult diets" (Idea: Imaging of bacteria and insects; 11. Research carried out by marking and tracking bacteria). contract and agreement holders. 11. Reports and publications. 11. Renewal requests and continued funding of RCM’s and 11. Continue R&D. CRP. 12. Final RCM held in 2017. 12. Final CRP report completed. 12. Research and dissemination activities concluded. 12. Hold final RCM to review data and synthesize results (early 13. CRP evaluation carried out. 2017). 13. CRP evaluation report 13. CRP evaluation by Agency completed. committee is positive. 13. Evaluate the CRP and submit 14. CRP members submit papers evaluation report. summarizing activities. 14. Peer-reviewed manuscripts

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14. Publications in scientific published. 14. Summarize and publish literature. advances of CRP in a special issue of a peer-review journal.

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ANNEX 1 (List of participants)

Second RCM on “Use of Symbiotic Bacteria to Reduce Mass-rearing Costs and Increase Mating Success in Selected Fruit Pests in Support of SIT Application” 6-10 May 2014, Bangkok, Tahiland ARGENTINA BRAZIL Mr Segura Diego Fernando Mr Fernando L. Consoli Instituto Nacional de Tecnología Escola Superior de Agricultura Agropecuaria “Luiz de Queiroz”, Avenida Padua Dias 11 Casilla de Correo 25, Los Reseros y Caixia Postal 9 Las Cabañas s/n 13418-900 Piracicaba, S.P. 1712 CASTELAR, PCIA. DE BUENOS Tel: +55 19 342941 AIRES Email: [email protected] ARGENTINA Email: [email protected] Ms NIU Changying College of Plant Science and Technology AUSTRALIA Huazhong Agricultural University Mr Peter Crisp Rd. Shizishan #1 South Australian Research and WUHAN HUBEI 430070 Development CHINA Institute (SARDI) Email: [email protected] Gate 2b, Hartley Grove GPO Box 397 CHILE Adelaide, SA 5001 Ms Carolina Yañez Prieto Tel: 61 8 8303 9371 Instituto de Biología, Facultad de Ciencias Email: [email protected] Pontificia Universidad Católica de Valparaíso Ms Olivia Reinolds Av. Universidad 330, Curauma School of Agricultural and Wine Sciences, Valparaíso Charles Sturt University Email: [email protected] Editorial Board Member, Insects Woodbridge Road, Menangle NSW 2568, GREECE Australia Mr George Tsiamis Private Bag 4008, Narellan NSW 2567, University of Western Greece Australia 2 Seferi Street W:+61 (0) 2 4640 6426 30100 Agrino M: +61 (0) 438276803 Email: [email protected] F: +61 (0) 24640 6300 Email:[email protected] Mr Nikolaos Papadopoulos School of Agricultural Sciences University of Thessaly BANGLADESH Fytokou Street Ms Mahfuza Khan 38446 Volos N. Ionia Bangladesh Atomic Energy Commission Tel: 30 2421093285 (BAEC) Email: [email protected] E-12/A, Agargaon, sher-e-Banglanagar Dhaka 1207 Tel : +880 2 778 9423 Email: [email protected]

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Mr Ntougias Spyridon MEXICO Democritos University of Thrace Mr Hemilio Hernandez Vas. Sofias Str. 12 Secretaria de Agricultura, Ganaderia, 67100 XANTHI Desarrollo Rural, Pesca y Alimentacion GREECE SAGARPA Email: [email protected] Carretera Tapachula-Cd. Hildago, km 19.5 30860 Metapa, Chiapas GUATEMALA Tel: 52 962 6435059 Ms Dyna Melgar Email: [email protected] Programa MOSCAMED Ministerio de Agricultura, Ganaderia y SPAIN Alimentacion (MAGA) Mr Jaime Garcia de Oteyza 16 calle 3-38, Zona 10 Centro de Control Biologico de Plagas Ciudad de Guatemala 01010 Poligono 9, Parcela 13, Paraje la Cantina Tel: 502 77402900 Ext 118 46315 Caudete de las Fuentes Email: [email protected] Tel: 34 962319515 [email protected] Email: [email protected]

INDIA TURKEY Mr Hire Ramesh Mr Ayidin Suzu Tuncbilek Bhabha Atomic Research Centre (BARC) Erciyes University Faculty of Sciences Trombay Dept. of Biology Mumbai, Maharashtra 400 085 38039 Kayseri Tel: + 91 22 2559 2292 Tel: 90 352 4374901 Email: [email protected] Email: [email protected]

ISRAEL UNITED STATES OF AMERICA Mr Ben-Yosef Michael Ms Mateos Mariana Hebrew University of Jerusalem Texas A&M University (TAMU) Mount Scopus P.O. Box 3473 TAMU, Teague Research 91905 JERUSALEM Center ISRAEL COLLEGE STATION, TX 77843 Email:[email protected] UNITED STATES Email: [email protected] ITALY Ms Patrizia Sacchetti Ms Erin Schuenzel Dipartimento di Biotecnologie Agrarie University of Texas-Pan American (DIBA) 1201 W University Drive Universita degli Studi die Firenze Edinburg, TX 78539 Via Maragliano 77 Tel: 956 665 2229 50144 Email: [email protected] Tel: 30 26410 74114 Email: [email protected] IAEA Scientific Secretary Mr Carlos Caceres Insect Pest Control Laboratory FAO/IAEA Agriculture & Biotechnology Laboratories 2444 Seibersdorf, Austria Email:[email protected]

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OBSERVERS ISRAEL Mr Boaz Yuval BELGIUM Hebrew University of Jerusalem Dept. of Mr Virgilio Massimiliano Entomolgy Royal Museum for Central P.O. Box 12 Rehovot 76100 Africa Joint Experimental Tel: 972 2 6586638 Molecular Unit (JEMU) Email: [email protected] Leuvensesteenweg 13 3080 TERVUREN BELGIUM Mr Jurkevitch, Edouard Email:massimiliano.virgilio@a Department of Entomology fricamuseum.be Hebrew Universiy of Jerusalem P.O. Box 12, 1 Hertzl Street 76100 REHOVOT BRAZIL ISRAEL Ms Aguiar Jordao Paranhos Beatriz Email: [email protected] Avenida Brazil 879 CEP Mr Zaada Doron 13416-530 PIRACICABA Department of Entomology SAO PAULO Hebrew University of Jerusalem BRAZIL Email: [email protected] Email: [email protected]

CHINA ITALY Mr Dong Yongcheng Ms Bigiotti Gaia College of Plant Science and Technology Department of Agrifood Production and Huazhong Agricultural University Environmental Sciences Wuhan Section of Plant Pathology and HUBEI PROVINCE 430070 Entomology CHINA Via Maragliano Email: [email protected] 77 - 50144 FIRENZE ITALY Email: [email protected]

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ANNEX 2 (Agenda)

The Use of Symbiotic Bacteria to Reduce Mass-Rearing Costs and Increase Mating Success in Selected Fruit Pests in Support of SIT Application Fourth Research Coordination Meeting - organized by the Joint FAO/IAEA

Vienna International Centre Vienna, Austria, 17-21 May 2017

Room MOE79 (on 17 May 2017); Room M5 (from 18-21 May 2017 inclusive). Side rooms: MOE05 and MOE07) AGENDA WENSDAY, 17 MAY 2017 08:45 – 09:00 Carlos Caceres: Welcome statement, administrative issues and goals of the meeting. SESSION I - Chairperson: Erin Schuenzel

09:00 – 09:30 George Tsiamis et al - Greece: Characterization of SymBioKosmos of Bactrocera dorsalis complex of fruit flies 09:30 – 10:00 M. Aceituno-Medina & E. Hernández - Mexico: Progress of the role of the associated bacteria on the biology, rearing and behavior of Anastrepha obliqua, A. serpentina and A. striata and other topics.

COFFEE BREAK (Bank issues)

10:30 – 11:00 Diego Segura et. al. - Argentina: Bacteria associated to Anastrepha fraterculus (Diptera: Tephritidae): characterization and effect on sterile males sexual competitiveness. 11:00 – 11:30 Boaz Yuval, Michael Ben-Yosef, Doron Zaada, Maayan Shamir & Edouard Jurkevitch - Israel: Fruit, Flies & Bacteria: Insights from the Medfly and the Olive Fly 11:30 – 12:00 Spyridon Ntougias et al. – Greece: Cultivation of the medfly gut symbiont Providencia sp. AA1 in bioreactor systems for mass rearing and sterile insect technique applications

LUNCH

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SESSION II - Chairperson: Boaz Yuval

13:30 -14:00 Erin Schuenzel, et al. - USA: Identification of bacteria with detrimental and beneficial effect on the larval survivorship of the Mexican fruit fly (Anastrepha ludens) 14:00–14:30 Dyna Melgar - Guatemala: Typification of microorganisms at Emergence and Release Facilities that use genetic sexing strains of Ceratitis capitata and Anastrepha ludens 14:30 – 15:00 Carolina Yañez Prieto - Chile: Characterization of total and diazotrophic gut bacterial communities in the Mediterranean fruit flies Ceratitis capitata (Diptera: Tephritidae) subjected to sterile insect technique

COFFEE BREAK

15:30 – 16:00 Teresa Navarro, Jaime García de Oteyza - Spain: Evaluation of main quality parameters in Mass rearing and Release Facilities associated with the use of probiotics

16:00 – 16:30 Changying Niu et al.- China: Identification and manipulation of symbiotic bacteria associated with Bactrocera dorsalis and Bactrocera minax

16:30 – 17:00 Malini Alleck et al. – Mauritius: Impact of Klebsiella oxytoca on quality of Bactrocera zonata and Zeugodacus cucurbitae

THRUSDAY, 18 MAY 2017

SESSION III - Chairperson: Diego Segura 08:30 – 09:00 Peter Crisp - Australia: The physiological importance of bacteria isolated from the gut of Dirioxa pornia (Tephritidae) and the relationship with alimentary canal and the structure of the oesophageal diverticulum 09:00 – 09:30 Ramesh S. Hire and Ashok B. Hadapad - India: Integration of sterile insect technique and symbiotic bacteria for the control of Bactrocera cucurbitae 09:30 – 10:00 Mahfuza Khan et al. – Bangladesh: Exploitation of gut microbial community on adult fitness and larval rearing of the melon fly, Bactrocera cucurbitae (Coq.) and the oriental fruit fly, Bactrocera dorsalis (Hendel) in support of SIT application

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COFFEE BREAK

10:30 – 11:00 Ayidin Suzu Tuncbilek - Turkey: Wolbachia-based Strategies to Increase Sterile Ceratitis capitata Wiedemann (Diptera: Tephritidae) Quality in Support of SIT 11:00 – 11:30 Nikos Papadopoulos et al. - Greece: Development of Wolbachia infected medflies in different temperatures regimes and host fruits and effect of exposure to orange oil on male mating competitiveness and the performance of the endoparasitoid Aganaspis daci 11:30 – 12:00 Patrizia Sacchetti et al. - Italy: Practical implication of the olive fly symbiosis

LUNCH

13:30 -14:00 O.L. Reynolds et al.- Australia: Larval Gut Microbiota of Bactrocera tryoni Froggatt (Diptera: Tephritidae) and the effects of bacterial probiotics fed to larvae of Queensland fruit fly (Bactrocera tryoni): do they improve insect quality under the Sterile Insect Technique? SESSION V Presentations Discussion - Chairperson: Oliva Reynolds 14:00 – 14:30 All participants

SESSION VI: Background situation analysis, baseline knowledge from the last RCM, progress made, outcomes achieved and identification of gaps that need to be addressed - Chairperson: Carlos Caceres and Group Leaders

14:30 – 17:30 Working Groups: Background situation analysis, baseline knowledge from the last RCM, progress made, outcomes achieved and identification of gaps that need to be addressed.

FRIDAY, 19 MAY 2017

08:30 – 10:00 Working Groups: Background situation analysis, baseline knowledge from the last RCM, progress made, outcomes achieved and identification of gaps that need to be addressed.

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COFFEE BREAK 10:30 – 12:00 General Discussion: Review baseline knowledge at start of CRP, progress made and outcomes achieved during the CRP; Agreement on content of RCM report, drafting and compiling of RCM report LUNCH 13:30 – 17:30 Preparation of the final report of the CRP by the participants in different working groups. SATURDAY, 20 MAY 2017 08:30 – 12:00 Preparation of the final report of the CRP by the participants in different working groups. LUNCH

13:30 – 17:00 Preparation of the final report of the CRP by the participants in different working groups. 18:30 Dinner at 7Sternbräu (list for the confirmation of your participation will be circulated) SUNDAY, 21 MAY 2017

08:30 – 10:00 Finalization of the report COFFEE BREAK 10.30 – 12:00 Presentation of the final CRP report 12:00 – 14:00 Discussions on final papers for the special issue in the BMC Microbiology and closure of the RCM

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ANNEX 3 (Working groups)

1. Larval diets and radiation 3. Symbionts and novel 2. Probiotics effects control tools Erin Schuenzel* Emilio Hernandez Nikos Papadopoulos* Carolina Yanez Marina Mateus – H. Mariana Mateos, Dina Melgar Diego Segura Diego Segura Jaime Gaarcia Mshfuza Khan G. Tsiamis Emilio Hernandez Ramesh Hire Seibesdorf Mahfuza Khan Patrizia Sacchetti Olivia Reynolds, Peter Crisp Changying Niu Oliva Reynolds Nikos Papadopoulos* George Tsiamis* Peter Crisp Aydin Tuncbilek Peter Crisp Carlonia Yanez R. Hire – Ashok Hadapad Seibesdorf Jaime Garcia George Tsiamis Changgying Niu Spyridon Ntougias Boaz Yubal* George Tsiamis Erin Schuenze*l Dina Melgar Seibesddorf Spyridon Ntougias Rodolfo Munoz *Group leader

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ANNEX 4 (Abstracts of presentations)

TITLE OF WORKING PAPER: Bacteria associated to Anastrepha fraterculus (Diptera: Tephritidae): characterization and effect on sterile males sexual competitiveness.

AUTHOR (S): Segura, Diego F1,2; Conte, Claudia A.1; Juárez, M. Laura2,3; Pimper, Lida E.1; Salgueiro, Julieta; Cladera, Jorge L.1; Vera, M. Teresa2,3; Bourtzis, Kostas4, Tsiamis, George5; & Lanzavecchia, Silvia B.1.

ORGANIZATION: 1Instituto de Genética Ewald A. Favret (IGEAF), Instituto Nacional de Tecnología Agropecuaria (INTA), Castelar, Argentina; 2Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET); 3Cátedra Terapéutica Vegetal, Facultad de Agronomía y Zootecnia (FAZ), Universidad Nacional de Tucumán (UNT), Argentina; 4Insect Pest Control Laboratory, Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, Vienna, Austria; 5Department of Environmental and Natural Resources Management, University of Patras, Greece. SHORT SUMMARY OF PAPER Abstract: The association between bacteria and Tephritidae fruit flies has been known for more than a hundred years. However, the complexity of such associations and the availability of appropriate methods to characterize bacterial diversity caused several decades of slowdown. Molecular tools have significantly improved our ability to unravel such complex interactions which provoked a burst in the scientific literature over the last 10 years. A wide range of taxonomic groups of bacteria have been identified in association with Tephritidae fruit flies. These include bacteria living in rather lax association with their hosts as well as species located in specific structures or even endosymbiont bacteria, located inside the hosts’ cells. Likewise, a whole range of effects have been described, from pathogenic to beneficial, including modifications on behavioral, physiological and nutritional parameters. In this presentation we will summarize our most significant advances in the characterization of bacteria associated to Anastrepha fraterculus. The focus will be placed on gut bacterial community and the endosymbiont bacterium Wolbachia pipientis. In the first case, we will present results on: 1) the effect of antibiotic treatment on gut bacteria diversity and the fitness of the fly; 2) the characterization of isolate from wild and laboratory flies and their potential use as probiotics; 3) the effect of colonization of a wild population on the gut bacteria diversity. In the case of Wolbachia, we will summarize the results of our research aimed at describing the strains present in A. fraterculus sp. 1 from Argentina and the potential phenotypes these bacteria induce of their host.

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TITLE OF WORKING PAPER: Larval Gut Microbiota of Bactrocera tryoni Froggatt (Diptera: Tephritidae)

AUTHOR (S): A.T. Deutscher, (1,2), C. Burke (3), A. Darling (3), M. Riegler (4) O.L. Reynolds (1,2)# and T.A. Chapman (1)#

ORGANIZATION: (1) Biosecurity and Food Safety, NSW Department of Primary Industries, Elizabeth Macarthur Agricultural Institute, Menangle, New South Wales, Australia 2568, (2) Graham Centre for Agricultural Innovation (an alliance between NSW Department of Primary Industries and Charles Sturt University), Elizabeth Macarthur Agricultural Institute, Menangle, New South Wales, Australia 2568; (3) The ithree Institute, University of Technology Sydney, Sydney, New South Wales, Australia 2007; (4) Hawkesbury Institute for the Environment, Western Sydney University, Richmond, New South Wales, Australia 3121. # Dual senior authors

SHORT SUMMARY OF PAPER Abstract: The Queensland fruit fly, Bactrocera tryoni Froggatt (Diptera: Tephritidae) is native to Australia and is a biosecurity threat to our $9billion horticultural industry. This pest is controlled using a range of tools including the Sterile Insect Technique (SIT). The SIT involves the area-wide, inundative release of sterile insects to reduce reproduction in a wild population of the same species. Mass-rearing and irradiation (to render flies sterile) affects the quality of the released sterile insects. The fruit fly gut microbiota may influence insect health, fitness and behaviour. It is feasible that domestication, mass-rearing and irradiation impact an insect’s gut microbiota and affect the quality of mass- reared fruit flies used in SIT programs. To determine the difference in bacterial abundance and diversity of larval B. tryoni, we isolated the midgut from three domesticated colonies (reared on the same artificial carrot-based diet) and two wild populations (from peaches). We characterised the bacterial community from individual larval midguts, using near full-length (>1300 bp) 16S rRNA gene amplicon next-generation sequencing and culture-dependent methods. To determine the presence of, and identify yeasts, we isolated the midgut of larval B. tryoni, collected from a range of fruits and cultured the contents. Yeast-like isolates were randomly selected for analysis of the ITS1, 5.8S rRNA gene, and ITS2 sequences. Overall, larval gut bacterial diversity was low; however, wild larvae had greater diversity than domesticated larvae. A single bacterial genus, Asaia (Acetobacteraceae), not often detected in adult tephritids, was detected in all larvae (except one), suggesting these bacteria may have a fundamental role in B. tryoni larval development. Enterobacteriaceae and Leuconostocaceae were detected in wild larvae, but were largely absent from domesticated larvae. Larvae feeding on the same diet have a similar gut bacterial profile. However, larvae from different individual whole fruit, on the same tree, may have a different gut bacterial profile. Yeasts and yeast-like fungi of the genera Aureobasidium, Candida, Cryptococcus, Hanseniaspora, Pichia, and Starmerella were identified. Consuming yeasts could be nutritionally beneficial to the larvae, but are largely overlooked in tephritid gut microbiome work, and should be considered alongside bacteria. Ultimately, an understanding of the gut microbiota, i.e. both yeast and bacteria, of larval tephritids could lead to improved diets for mass-reared flies, resulting in reduced mass-rearing costs and an increase in the quality of tephritids used in SIT programs.

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TITLE OF WORKING PAPER: The effects of bacterial probiotics fed to larvae of Queensland fruit fly (Bactrocera tryoni): do they improve insect quality under the Sterile Insect Technique? AUTHOR (S): Shuttleworth, L.A. (1), & Reynolds, O.L. (1,2)

SHORT SUMMARY OF PAPER Abstract: The Queensland fruit fly, Bactrocera tryoni (Diptera, Tephritidae) is the most significant endemic biosecurity threat to Australia’s $9 billion horticultural industry. Bactrocera tryoni is controlled using a range of tools including the Sterile Insect Technique (SIT). SIT involves area-wide, inundative releases of sterile insects to reduce reproduction in a wild population of the same species. The provision of bacterial probiotics to larval B. tryoni may improve the reduced quality of mass-reared flies in SIT programs. Bacteria from the genera Asaia, Enterobacter, Lactobacillus, and Leuconosto were isolated from the midguts of wild larvae and provided as live bacterial probiotic supplements (individually and a blend) in the diet of mass-reared larvae. Several insect quality traits were tested including larval development time, time to adult eclosion, sex-ratio, flight ability, pupal weight, locomotor activity, and field cage survival. Enterobacter reduced both larval development time, and time to adult eclosion, while Leuconostoc and Lactobacillus delayed larval development time, and time to adult eclosion, compared to the control, i.e. diet without probiotic supplementation. The blend and Asaia treatments were similar to the control for adult emergence, yet Asaia reduced, and the blend delayed larval development time. Higher adult male sex ratios were produced by the blend, Asaia and Leuconostoc (1.5:1, 1.4:1, 1.3:1, male:female respectively). All probiotic treatments had reduced pupal weight. Enterobacter and Asaia had higher total locomotor activity, while Lactobacillus, Leuconostoc and the blend all had lower activity, compared with the control. Preliminary data show decreased adult survival of Enterobacter fed larvae. The results suggest that bacterial probiotics fed to larvae influence a range of larval and adult quality traits. The provision of probiotics to larval B. tryoni may have application in SIT operational programs.

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TITLE OF WORKING PAPER: “The physiological importance of bacteria isolated from the gut of Dirioxa pornia (Tephritidae) and the relationship with alimentary canal and the structure of the oesophageal diverticulum”

AUTHOR (S): Dr Peter Crisp, Kala Bhandari

ORGANIZATION: South Australian Research and Development Institute SHORT SUMMARY OF PAPER Abstract: Supplementing the diet of adult Tephritid fruit fly species Bactrocera tryoni and Dirioxa pornia with selected enterobacteria isolated from the gut of wild D. pornia was demonstrated to increase fecundity and pupal emergence (Bhandari, Crisp 2014). The initial phase of research was to identify dacteria from the gut of adult D. pornia. Through analysis of the 16S rRNA gene utilising PCR (Polymerase Chain Reaction) and sequencing of individual culturable bacteria. To get a broader profile of the entire bacterial community, the whole gut was sequenced. Regardless of method used the majority of bacteria identified belonged to two orders, Lactobacillales (Firmicutes: Bacilli) and Pseudomonadales (Gammaproteobacteria: Proteobacteria). To identify the bacteria associated with the improved reproduction success diet supplementation trials were conducted with culturable Enterobacteria isolated from the gut of D. pornia. These bacteria induced similar changes improvement in B. tryoni fecundity as was seen for D. pornia and suggests that they work as a probiotic. In order to better understand the relationship between the Tephritid flies and these bacteria Micro computed tomography techniques were used to conduct a virtual dissection of the adult fly to locate the alimentary tract, especially the oesophageal diverticulum. To improve the understanding of this relationship is the isolation and identification of bacteria in the oesophageal diverticulum, crop, midgut and reproductive system using nextgen sequencing are being conducted. Micro CT scan imaging is also being undertaken on B. tryoni.

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TITLE OF WORKING PAPER: Exploitation of gut microbial community on adult fitness and larval rearing of the melon fly, Bactrocera cucurbitae (Coq.) and the oriental fruit fly, Bactrocera dorsalis (Hendel) in support of SIT application

AUTHOR (S): 1Mahfuza Khan, 1Kajla Seheli, 1M. Abdul Bari, 1Shakil A. Khan, 2GeorgeTsiamis and 3Kostas Bourtzis

ORGANIZATION: 1 Insect Biotechnology Division, Institute of Food and Radiation Biology Atomic Energy Research Establishment, Ganak Bari-1349, Savar, Dhaka, Bangladesh 2Department of Environmental and Natural Resources Management University of Patras 3Insect Pest Control Laboratory, Joint FAO/IAEA Programme of Nuclear Techniques in Food and Agriculture, Vienna, Austria.

SHORT SUMMARY OF PAPER Abstract:

Sterile Insect Technique (SIT) is one of the proven control methods of Area-Wide Pest Management Programme of Tephritid fruit flies worldwide. Low cost mass rearing facilities and production of high quality insect pests are major components of any SIT programme. Tephritidae is a large family that includes many fruit pests which are usually adopted for housing large quantities of bacteria in their digestive tract. Explorations on the bacterial community of different life stages (eggs, larvae, and adults) of the Melon fly, Bactrocera cucurbitae (Coq.) and the Oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera:Tephritidae) was determined using cultural, morphological, bio-chemical tests, and API kits. The mid gut bacteria community of B. cucurbitae and B. dorsalis also determined using molecular approaches like 16S rRNA, 16S pyrotagging and phylochip approaches. Observation on the possible change of gut microbial community of irradiated and control B. cucurbitae and B. dorsalis was also evaluated using above mentioned methods. Selected isolated gut bacterial species were further examined by incorporated into protein (casein:yeast extract:sugar, 1:1:2) and sugar diets to study the fitness parameters, viz., fecundity, longevity and mating successes of B. cucurbitae and B. dorsalis under laboratory and semi-field cage study. To improve the quality parameters of B. dorsalis developed from laboratory established liquid and gel based artificial larval diets several gut bacteria spp., Enterbacter cloacae, Pantoea spp 3, Proteus mirabilis, Serratia spp., Staphylococcus haemolyticus was tested. The prevalence of reproductive parasite, Wolbachia was determined for four Bactrocera spp. B. cucurbitae, B. dorsalis, Bactrocera zonata (Saunders), and Bactrocera tau (Walker) collected from different areas of Bangladesh. Based on different colony morphology total forty and thirty five predominant gut-bacterial isolates were selected from the adult B. cucurbitae and B. dorsalis, respectively. The bacterial species viz., Aeromonas hydrophila-1, Aeromonas hydrophila- 2, Alcaligenes faecalis-1, Alcaligenes faecalis-2, Enterobacter aerogenes, Klebsiella oxytoca, Serratia mercescens, Serratia liquefaciens and Streptobacillus moniliformis was identified from the gut of B. cucurbitae. Whereas Aeromonas hydrophila-1, Aeromonas hydrophila-2, Aeromonas hydrophila-3, Bacillus alvei, Erwinia herbicola, Moraxella

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lacunata, Serratia liquefaciens, Serratia rubidaea, Streptobacillus moniliformis, Proteus inconstses, Proteus rettgeri, and Proteus vulgaris was identified from B. dorsalis. Using API 20E and API 20NE methods total fifteen genera and twenty one species of bacteria were identified from eggs and the mid-gut of larvae and adults of B. cucurbitae and B. dorsalis. The common bacterial spp. was Enterobacter cloacae, Delftia acidovorans, Pseudomonas putida and Stenotrophomonas maltophilia. The gut bacterial community of the larvae of both B. cucurbitae and B. dorsalis was 50 % similar irrespective of different rearing media. The effect of bacteria on different fitness parameters of B. cucurbitae and B. dorsalis found to be diet-dependent. The mating performance of B. dorsalis fed on K. oxytoca and P. rettgeri added protein and sugar diets appeared promising, but not for B. cucurbitae. Adult fly’s gut bacteria spp., Erwinia herbicola and Serratia sp. help to enhance mating success in terms of early mating of B. cucurbitae and B. dorsalis when incorporated with sugar diet compared to those fed on only sugar diet. E. herbicola and Serratia sp. enriched protein and sugar diets showed no significant effect on the ovariole number of B. cucurbitae and B. dorsalis after 14 days of adult emergence. However, delayed ovariole development and egg production were observed between 24-33 days when fruit flies fed on E. herbicola and Serratia sp. enriched sugar diets. Whereas, no egg production was recorded for both the fly species fed on only sugar diet. Mortality rate of B. cucurbitae and B. dorsalis fed on P. rettgeri, K. oxytoca, E. herbicola and Serratia sp. enriched sugar diets was higher than the bacteria enriched protein diets. No significant difference was observed between mean mortality of control and sterile B. dorsalis fed on different diet treatments under semi-field cage experiment. Significantly better quality parameters in terms of % egg hatch (88±52 %), mean number of pupae (3008±541), % flight ability (73±5%) were recorded for B. dorsalis than other bacteria added diets and control diet while active Serratia sp. added to liquid larval diet. In a separate experiment enrichment of gel based larval diet incorporated with larval gut bacteria sp. Enterobacter sp. showed improved adult emergence (92.7%), flight ability (87.0 %), and % survival (83.3%) under stress than control diet without affecting egg hatch, pupal yield, pupal weight, and larval duration of B. dorsalis. The prevalence of Wolbachia infections differed significantly among various populations of the Bactrocera complex of Bangladesh. Higher level of Wolbachia infections were detected for population of B. dorsalis, followed by B. zonata, B.cucurbitae, and B. tau. The presence of three new allelic profiles or Sequence Types (ST) in the Wolbachia strains was determined from the Bactrocera populations examined so far. A total of 8 WSP HVR profiles were identified with all of them being new in the Wolbachia WSP database.

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TITLE OF WORKING PAPER: Characterization of total and diazotrophic gut bacterial communities in the Mediterranean fruit flies Ceratitis capitata (Diptera: Tephritidae) subjected to sterile insect technique AUTHORS: Carolina Yáñez1, Natali Zamora1, Constanza Rojas1, Mauricio Núñez1, Mayerling Díaz2 and Hernán Donoso2 ORGANIZATION: 1Instituto de Biología, Facultad de Ciencias, Pontificia Universidad Católica de Valparaíso, Valparaíso, Chile; 2Centro de Producción de Insectos Estériles (CPIE), Servicio Agrícola y Ganadero, Región Arica y Parinacota, Chile.

SHORT SUMMARY OF PAPER Abstract: The Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), is one of the most serious agricultural pests in the world. Chile was declared a fruit-fly free country in 1995 and the sterile insect technique (SIT) was the key to the successful eradication. In this technique, mass- reared δ-irradiated male insects are released into the field where they compete with wild males for copulations with wild females. However, sterile medfly males show reduced sexual competitiveness and longevity, and it is likely that these effects could be caused by the disruption of the gut microbiota through the irradiation processes.

During this project, we aimed to characterize and identify the intestinal microbiota associated with C. capitata (Wied.) at different life stages under mass rearing conditions (injection and release streams), including a closer examination of the diazotrophic bacterial community. Additionally, we analyzed the effect of irradiation on bacterial populations in adult male flies and studied the structure of bacterial communities in wild specimens obtained from Argentina. We used 16S rRNA gene sequencing for the identification of bacterial isolates and denaturing gradient gel electrophoresis (DGGE) to study the diversity of total (16S rRNA gene) and diazotrophic bacterial communities (nifH gene) in mass-reared medflies.

We found that members of the family Enterobacteriaceae constitute the dominant populations in the gut of C. capitata. Isolates belonging to genera Serratia, Citrobacter, Morganella, Enterobacter, Providencia, Klebsiella were found in pupae and adults. Appearance of Stenenotrophomonas sp. and Pseudomonas sp. was observed after radiation. Culturable bacterial populations were affected by radiation, being Morganella and Providencia major bacterial genera absent in male sterile flies. Morganella and Providencia isolates were used for the evaluation of bacterial viability on adult diet, showing a very limited persistence (only 2 days). We also test the effect of bacteria-enriched diet (Enterobacter, Providencia, or Klebsiella) on sterile male survival in the laboratory, observing some differences in the survival when using Klebsiella.

DGGE results showed that the structure of bacterial communities (total and diazotrophic) associated with C. capitata vary in the different developmental stages. DGGE results showed that radiation do influence the composition of the intestinal microbiota, introducing changes in species richness but not affecting the diversity. However, no significant differences in diazotrophic species richness in irradiated and non-irradiated individuals were observed. Our results suggest that the composition of the gut bacterial communities in C. capitata is variable and changes in this variability cannot be only attributed to the effect of the radiation.

Finally, we analyzed the total and diazotrophic bacteria associated with wild C. capitata flies. Bacterial communities showed a simple structure and share some similarities with communities from mass-reared flies. Among diazotrophs, we isolated Klebsiella sp., as found in mass-reared

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flies, and Kluyvera ascorbata, not reported among isolates from mass-reared flies analyzed during this project.

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TITLE OF WORKING PAPER: Identification and manipulation of symbiotic bacteria associated with Bactrocera dorsalis and Bactrocera minax

AUTHOR (S): Awawing A. Andongma, Yulei Wang, Lun Wan, Xueping Dong, Adnan Mohamad, Kanjana Khaeso, Changying Niu

ORGANIZATION: College of Plant Science & Technology, Huazhong Agricultural University, Wuhan 430070, China

SHORT SUMMARY OF PAPER Abstract: Background: Insects are associated with bacteria that may affect their fitness in diverse ways; for example bacterial symbionts may play vital nutritional roles in insect larval development and adult reproduction. In recent years, the intricate relationship between fruit flies and their symbiotic bacteria has gained global attention. In this study we researched Bactrocera dorsalis (Hendel) and B. minax (Enderlein) (Diptera: Tephritidae) to understand their bacterial community structures, the effects of bacteria on growth and fitness parameters of the flies, and the transmission pattern by which bacteria are moved across the life cycle of flies. The results provide a better understanding of the insect-bacteria interaction and pave the way for the development of efficient and sustainable pest management programs.

Methods: Culture dependent and pyrosequencing techniques were used to identify the bacteria communities associated with B. dorsalis and B. minax. Antibiotics were provided and supplement of isolated bacteria in the diets to assess the impacts of bacteria on fly and larval fitness. Transmission patterns of bacteria were investigated using GFP fluorescent marker.

Results: In B. minax, 162 bacterial operational taxonomic units (OTUs) were identified at a 97% similarity interval. Proteobacteria was the dominant phyla representing the most abundant reads (80%) in all life stages. There was a shift in the most abundant reads from the early to the late developmental stages and the adult stages. The bacteria which became more prevalent in late juvenile and adult stages were Klebsiella pneumoniae, Providencia rettgeri and Enterobacter aerogenes. Feeding antibiotics to B. minax significantly decreased fecundity; while in contrast flies fed diets supplemented with K. pneumonia had significantly enhanced fecundity. Similarly, when flies were fed with Citrobacter baarkii, the mean number of copulations of male flies significantly increased. These results suggest that gut resident bacteria improve B. minax’s fitness.

From wild populations of B. dorsalis, high-throughput results showed that total reads could be assigned to 172 OTUs belonging to six phyla. Proteobacteria again dominated in immature stages, while Firmicutes dominated in adults. The most abundant families were Enterococcaceae and Comamondaceae. After bacterial transformation with GFP fluorescent proteins, Enterobacter cloacae and Enterococcus faecalis formed bacterial films in B. dorsalis gut 48 hours after ingestion. Additionally, E. faecalis administered to B. dorsalis eggs were monitored to be present throughout the life cycle, indicating the possibility of vertical transmission in B. dorsalis. In a long term cultured genetic sexing strain of B. dorsalis, isolated bacteria belonged to four phyla: Firmicutes, Proteobacteria, Bacteroidetes and Actinobacteria. The response of B. dorsalis to different gut isolates varied greatly. Diets enriched with Enterococcus phoeniculicola ended up with lower larval development duration, higher pupal weight, and increased survival.

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Conclusions: Symbiotic bacteria associated with B. minax and B. dorsalis play a vital role in their survival and reproduction which could be exploited in their sustainable management. The interaction between different fruit flies and their symbionts is complex and closely depends on hosts and other factors.

Keywords: Symbiotic bacteria, fitness, Bactrocera dorsalis, Bactrocera minax

This study was funded by International Atomic Energy Agency (via Research Contract CRP No. 17153) and the Joint National Science Foundation of China-Israel Science Foundation project (No. 31661143045).

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TITLE OF WORKING PAPER: Development of Wolbachia infected medflies in different temperatures regimes and host fruits and effect of exposure to orange oil on male mating competitiveness and the performance of the endoparasitoid Aganaspis daci

AUTHOR(S): Nikos T. Papadopoulos, Giorgos Kyritsis, Charalampos Ioannou, Niki Dionysopoulou and Kostas Zarpas

ORGANIZATION: Laboratory of Entomology and Agricultural Zoology, University of Thessaly SHORT SUMMARY OF PAPER Abstract: Recent studies have demonstrated that, further to inducing cytoplasmic incompatibility, Wolbachia infection affect immature survival and developmental duration as well as adult longevity and patterns of egg laying in trans-infected Mediterranean fruit flies (medflies), Ceratitis capitata (Diptera: Tephritidae). However, whether different host fruit species and temperature regimes affects immatures development of Wolbachia infected lines has not been explored yet. Likewise, minimal information exists regarding effects of exposure to orange oil (OO) on the mating competitiveness of Wolbachia infected males. Using the non-infected Benakeio medfly strain, the trans-infected lines Wol/Med 88.6 and Wol/Med S-10-3 that carry the Wolbachia strain and wCer2, and a wild medfly population obtained from field infested fruit we recorded the immatures developmental duration and survival in two host fruits (apples, bitter oranges) and three constant temperature regimes (18, 25 and 30 oC). In addition, using the same medfly populations and including both wCer2 and wCer4 Wolbachia infected lines we studied, under constant conditions (25 oC, 65%RH; 14:10 L:D) and following a two choice experimental design, the effects of OO exposure on male mating competitiveness. As it was expected results demonstrate that temperature is inversely correlated with immatures developmental duration. Development in bitter oranges is faster than in apples. The three different populations tested seem to respond similarly in terms of average immature developmental duration regardless of the host and temperature. Nonetheless, interesting “phenotypes” emerged when the variance and distribution of individual developmental duration is considered. Both the Benakeio and the wCer2 infected populations expressed low survival rates in both fruit species and most temperatures tested. It seems that the wCer2 infected flies suffer higher mortality rates in bitter oranges with inconsistent patterns emerging across the three different temperatures. Survival patterns of the wild population were low but consistent among the three temperatures in apples and quite high and consistent in bitter oranges. Exposure to OO had no effect on male mating competitiveness of either the Benakeio or the wCer2 and wCer4 infected males when laboratory adapted females of the same type were used. Mating rates were extremely low for both the Benakeio and wCer2 males and no firm conclusions can be extracted when wild females were considered. The wCer4, OO exposed males mated in lower frequencies with wild females compared to non-exposed ones. In another series of studies, we explored the effect of medfly Wolbachia infection on parasitism rates, immature developmental duration and adult longevity of the endoparasitoid Aganaspis daci. Wolbachia infected medfly pupae yielded fewer male parasitoids and increased the parasitoid immature developmental duration compared to non-infected pupae. Interestingly, A. daci females, but not males, emerging from Wolbachia infected medfly pupae expressed longer longevity than those obtained from non-infected pupae.

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TITLE OF WORKING PAPER: Cultivation of the medfly gut symbiont Providencia sp. AA1 in bioreactor systems for mass rearing and sterile insect technique applications

AUTHOR (S): Ioanna Zerva1, Konstantinos Azis1, Paraschos Melidis1, Carlos Caceres2, Kostas Bourtzis2 and Spyridon Ntougias1

ORGANIZATION: 1Laboratory of Wastewater Management and Treatment Technologies, Department of Environmental Engineering, Democritus University of Thrace, Vas. Sofias 12, 67100 Xanthi, Greece; 2Insect Pest Control Laboratory, Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, Vienna, Austria

SHORT SUMMARY OF PAPER Abstract: In order to elucidate the role of medfly-gut bacteria in the diet of insects used for mass rearing and sterile insect technique (SIT) applications, the symbiont Providencia sp. AA1 was previously isolated from the midgut of Ceratitis capitata at the Joint FAO/IAEA Insect Pest Control Laboratory and its positive effects on mass rearing and male mating competitiveness were assessed. However, lack of technologies to produce adequate biomass is the main limitation to scale-up applications of gut microbes. Thus, this work attempted to identify the growth pattern of Providencia sp. AA1 in laboratory-scale bioreactors for future biotechnological applications regarding sterile insect technique. Phylogenetic analysis revealed that strain AA1 was associated with the species Providencia rettgeri and P. vermicola. Providencia sp. AA1 could grow in both LB and cheese whey wastewater, which can be used as an alternative cheap carbon source to replace commercial media, exhibiting specific growth rates in batch reactors of 2.19 and 0.55 h-1, respectively. Based on the examination of its physiological characteristics, Providencia sp. AA1 can be characterized as a pectinase-negative mesophilic, halotolerant, facultatively anaerobic, neutrophilic bacterium, with broad pH range for growth. Strain AA1 could ferment glucose, hydrolyze urea and utilize citrate, being also capable of producing acetoin and giving a strong -1 -1 catalase reaction. The specific oxygen uptake rate of biomass was 38.89 ± 1.73 mg O2 g VSS h , with the yield coefficient (YH) in acetate being equal to 0.73 ± 0.01 g biomass/g substrate o consumed. The maximum specific growth rate (μmax) of strain AA1 cultivated in bioreactor at 35 C was 0.067 h-1. Protein content estimated as 53.2 ± 3.2% of the dry biomass. Moreover, greater endocellular -glucosidase activity was determined during growth of the strain AA1 in LB than in cheese whey wastewater (p<0.01), whereas endocellular protease and lipase activities were undetectable. The respective extracellular activities were negligible in both LB and cheese whey wastewater. In conclusion, the ability of strain AA1 to grow under versatile environmental conditions and low-cost substrates makes feasible a scale-up cultivation approach.

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TITLE OF WORKING PAPER: Characterization of SymBioKosmos of Bactrocera dorsalis complex of fruit flies

George Tsiamis1, Hlias Asimakis1, Vangelis Ntountoumis1, Antonios A. Augustinos2, Ramesh Hire3, Ashok Hadapad3, Mahfuza Khan4, Kostas Bourtzis2 1 Department of Environmental and Natural Resources Management, University of Patras, 2 Seferi str., 30100 Agrinio, Greece 2 Insect Pest Control Laboratory, Joint FAO/IAEA Programme of Nuclear Techniques in Food and Agriculture, Vienna, Austria 3 Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre (BARC), Trombay Mumbai, Maharashtra, 400 085, India 4 Insect Biotechnology Division, Institute of Food and Radiation Biology, Atomic Energy Research Establishment, Ganak bari- 1349, Savar, Dhaka, Bangladesh

Abstract The globally occurring Tephritidae family contains more than 4000 species, most of which are polyphagous. The Bactrocera dorsalis complex of the Tephritidae family (subfamily Dacinae) contains 75 described species, which are mainly endemic to Southeast Asia. Several species, however, have also been reported in Africa, Middle East, Australia, Papua New Guinea, Indian Ocean and South America. This complex, also known as the Oriental fruit fly complex, is considered one of the most damaging insect pests globally, being responsible for annual losses of billions of USD. Despite the important role insect symbionts can have in the biology, physiology, ecology and evolution of their hosts, only a limited number of studies dealt with the B. dorsalis complex. One of the aims of this study was to detect and genotype reproductive parasites in laboratory and natural populations of B. dorsalis sensu stricto, B. carambolae and populations from species outside the complex like B. zonata, B. cucurbitae, B. scutellaris, B. carambolae, B. correcta, and B. nigrofemoralis. This has been completed and presented during the previous meeting. A brief summary will be presented, with a focus on the putative Horizontal Gene Transfer events characterized in B. dorsalis and B. zonata. The symbiotic flora associated with the gastrointestinal tract of B. dorsalis sensu stricto, B. cucurbitae, B. carambolae, and B. zonata was examined using an Illumina amplicon sequencing approach. The results of sequencing barcoded PCR amplicons will be presented and the possible correlation between bacterial communities, developmental stages, age and sex will be discussed as well. Finally, using a similar approach, the effect of radiation was examined in B. cucurbitae under two diets bran and sweet gourd. Our analysis indicates that the irradiated B. cucurbitae bred on bran lack Raoultella and Citrobacter completely and have the highest abundance in Providencia. An increase in the relative abundance of Providencia seems to cause a decrease in the members of the Enterobacteriaceae. Correlations between irradiation treatment, diet, and developmental stages will be presented in detail.

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TITLE OF WORKING PAPER: Typification of microorganisms at Emergence and Release Facilities that use genetic sexing strains of Ceratitis capitata and Anastrepha ludens.

AUTHOR (S): Dyna Melgar Dorigoni

ORGANIZATION: MOSCAMED Program, El Pino Mass Rearing Plant, Guatemala

SHORT SUMMARY OF PAPER Abstract: The last phase of production of sterile males of C. capitata and A. ludens genetic sexing strains consists on the emergence and maturation of insects in structures designed for emergence/feeding insects by providing a liquid or an agar based diet, prior to being subjected to low temperatures (Chill Adult release technique) and being released by ground or air. The first microbiological monitoring of these adult diets was conducted at the Retalhuleu C. capitata release center, where genera of Klebsiella pneumoniae, Klebsiellla oxytoca and Serratia marcescens were identified and confirmed, from which the latter had been previously reported as being pathogenic to adult insects. At the Pino mass rearing facility, an Emergence and Release Facility (ERF) was recently inaugurated; This ERF operates on the bases of using the Chilled Adult Release Technique. The microbial loads and type of microorganisms were monitored for 5 days. From these counts, the prevalence of K. oxytoca was established. in 4 consecutive batches. The bacteria where present in the liquid diet itself as well as on the sponges and cotton cloths with which the liquid diet is transferred by capillarity. In a microbiological monitoring carried out in Mission TX to the A. ludens strain TBPS7 from the mass rearing of the species in San Miguel Petapa, it was observed through high resolution microscopy equipment that insects collected in the emergence area showed distended abdomens and sometimes accompanied by liquefied internal organs, which also involve decreases of longevity, probably mating propensity and early death. All of these parameters reduce the efficiency of the sterile insect technique (SIT). In this case, the presence of Morganella morganii was identified as the pathogen, which was also detected in the larval rearing process. Under aseptic and small-scale conditions, K. oxytoca and Morganella morganii isolated from the larval rearing process were both inoculated in the diet and adult emergence conditions were simulated. Insects were closely monitored by microscopy and the presence of gas in the abdomen was checked. To conclude, the results of these studies indicate the importance of the microbiological management at the ERF’s, having as base line the monitoring of contact surfaces, diet, cloths for water supply and/or liquid diet and the importance of choosing a symbiont that adapts to the mass rearing process.

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TITLE OF WORKING PAPER: Integration of sterile insect technique and symbiotic bacteria for the control of Bactrocera cucurbitae

AUTHOR (S): Ramesh S. Hire and Ashok B. Hadapad

ORGANIZATION: Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Trombay, Mumbai – 400085 INDIA SHORT SUMMARY OF PAPER Abstract: Fruit flies of Bactrocera species are major insect pests of fruits and vegetables in India and damage around 30-100% produce. Currently, various control methods including Sterile Insect Technique (SIT) are being used for management of fruit flies. However, fitness and competitiveness of sterile insects are influenced by various parameters like associated bacterial endosymbionts. During the IAEA-CRP, we studied bacterial endosymbionts associated with the melon fly, B. cucurbitae. We collected fruit fly samples from 28 locations from ten states of India during the years 2012-2017. Eight Bactrocera species were identified from collected samples. Samples of B. cucurbitae and B. dorsalis complex species were used for screening of Wolbachia and assessment of gut microbiota. Based on the 16S rRNA sequencing, the dominant bacterial communities belongs to Enterobacteriaceae family and among them Enterobacter, Klebsiella, Citrobacter, Bacillus, Providencia, Micrococcus and Staphylococcus are the major bacterial species. Some of the melon fly gut bacteria showed promise in attracting Bactrocera sp. adults when tested under field conditions. Microbial diversity of irradiated and non-irradiated melon fly was assessed by studying the V3-V4 region of the bacterial 16S rRNA gene using metagenomic analysis. The melon fly populations collected from different geographical regions of India showed an average 52% Wolbachia infection. Phylogenetic analysis suggested that Wolbachia Supergroup A is prevailing in Indian population of B. cucurbitae. Melon fly cultures with and without Wolbachia were established in laboratory and used for studying the incompatible insect technique (IIT). Also, sterility dose for melon fly was optimised and evaluated. In conclusion, various endosymbionts associated with melon fly populations from India were identified and these gut bacteria could be employed to improve sterile insects in successful implementation of SIT.

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TITLE OF WORKING PAPER: Fruit, Flies & Bacteria: Insights from the Medfly and the Olive Fly

AUTHOR (S): Boaz Yuval, Michael Ben-Yosef, Doron Zaada, Maayan Shamir & Edouard Jurkevitch

ORGANIZATION: Hebrew University of Jerusalem

SHORT SUMMARY OF PAPER Abstract:

Our presentation reviews work conducted during the CRP focussed on the relationship between fruit fly larvae, host fruit, and associated bacteria.

Work on the monophagous Olive fly has established that these flies are associated with a single, obligate bacterium, Candidatus Erwinia dacicola. The presence of these bacteria in larval intestinal caecae allows larvae to detoxify and develop in immature, green olives. The consequence of this relationship is the temporal expansion of olive fly activity, allowing them to evolve from a univoltine species developing on mature olives within a restricted time period, to multivoltinism, exploiting their host throughout its fruiting cycle.

Studies on the polyphagous medfly have dealt with the interactions between larvae and bacteria within host fruit. We have established the following: a. Female anti-microbial excretions shape to some extent the inoculum of bacteria available within the fruit. b. No single bacterial species seems to promote larval growth in infested fruit. Rather, the combined consortia of bacteria promote rot and larval development. c. Although larvae effectively disperse bacteria in the host, we found no evidence for larvae as agents of increase in bacterial abundance within fruit. d. Within infested fruit, there is horizontal transmission of bacteria between larvae.

Future work will extend these experiments, and focus on additional species.

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TITLE OF WORKING PAPER: Practical implications of the olive fly symbiosis

AUTHORS: Sacchetti P.1, Bigiotti G.1, Pastorelli R.2, Guidi R.1, Arnone S.3, Sasso R., Cristofaro M.3, Viti C.1, Giovannetti L.1, Belcari A.1

ORGANIZATIONS: 1DISPAA, University of Firenze, Via Maragliano, 77, 50144 Firenze Italy; 2CREA-ABP, Via di Lanciola, 12, 50125, Firenze, Italy; 3ENEA Centro Ricerche Casaccia, Via Anguillarese, 301, 00123 , Roma, Italy

SHORT SUMMARY OF PAPER Abstract: The presence of bacterial symbionts in the olive fruit fly, Bactrocera oleae, was highlighted more than one century ago and since, this species has probably being received more attention than other fruit flies. Although molecular biology techniques have been markedly improved the basic knowledge on bacterial symbioses, relationships between the olive fruit fly and bacteria, included Candidatus Erwinia dacicola, has not completely elucidated yet. During the five-years CRP, almost all the issues proposed in the project have been explored, some outcomes have been achieved while further investigations are needed in order to be finalized. Concerning basic information on bacterial ecology in the olive fly population, several wild and lab reared olive fly adults have been dissected and oesophageal bulbs have been tested for bacterial presence. Ca. E. dacicola was always evidenced in wild specimens, confirming that the symbiont can occur as two different lineages. In the olive fly lab strain reared on cellulose-based diet, Ca. E. dacicola has never retrieved, while different bacterial species have been identified. Among them, Morganella morganii was the predominant one. Investigations carried out on the vertical transmission of the symbiont Ca. E. dacicola in wild olive flies have shown how the usage of antimicrobial products or preservatives can be detrimental in the colony establishment process. As regards the development of probiotic diets, we proved that some culturable bacteria, frequently occurring in the olive tree phylloplane, can be supplied to olive fly adults producing some beneficial effects on the olive fly physiology. Nevertheless, present results are not consistent and need further research. Finally, despite Ca. Erwinia dacicola is considered as an obligate endosymbiont, attempts to transfer it from wild populations to lab reared specimens have been performed, using different symbiont sources. Preliminary tests were carried out also on possible effects of irradiation on the olive fly symbiont. A batch of wild olive fruit fly pupae was divided in three subgroups, two of them were irradiated with 100 and 150 Gy, 4 days before the emergence, while the third one was used as control: adults were dissected in order to highlight differences in bacterial load between irradiated and non-irradiated flies.

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TITLE OF WORKING PAPER: Impact of Klebsiella oxytoca on quality of Bactrocera zonata and Zeugodacus cucurbitae

AUTHOR (S): Malini Alleck, Preeaduth Sookar, Sabrina Dyall

ORGANIZATION: (i) Ministry of Agro Industry and Food Security, Agricultural Services, Entomology Division, Réduit, Republic of Mauritius

(ii) Faculty of Science, University of Mauritius, Réduit, Republic of Mauritius

SHORT SUMMARY OF PAPER Abstract: Fruit fly problems in Mauritius date back to the beginning of the 19th century. The peach fruit fly, Bactrocera zonata, and the melon fly, Zeugodacus cucurbitae are the most important species attacking fruits and cucurbits respectively. Growers rely mainly on broad-spectrum insecticides for fruit fly control hence causing harmful effects to human and animal health and to the environment. An integrated pest management approach is hence being implemented and includes an SIT component. However, irradiation significantly disrupts native microbiota in fruit fly gut, thereby reducing the sterile insects’ quality and eventually the efficacy of SIT. During this study, gut bacteria were isolated from B. zonata and Z. cucurbitae using culture dependent methods on selected growth media and identified by performing 16S rRNA. The gut bacteria identified included Citrobacter freundii, Citrobacter koseri, Enterobacter cloacae, Klebsiella oxytoca, Klebsiella pneumoniae, Pantoeaagglomerans, Providencia alcalifaciens and Providencia vermicola. One of the bacterial species was supplemented in the adult diet of B. zonata and Z. cucurbitae and assessed on a few fitness parameters. The impact of Klebsiella oxytoca on longevity, fertility and fecundity of Z. cucurbitae was evaluated. Flies fed on K. oxytoca survived longer than sugar fed flies. Fertility as well as fecundity of the females were higher as compared to untreated females. The effect of K. oxytoca was also evaluated on survival and mating competitiveness of sterile B. zonata. A lower mortality was observed with flies fed on bacteria and normal diet as compared to flies fed on sugar only. The number of matings of sterile males fed on normal diet and on bacteria was also higher as compared to sugar fed flies.

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TITLE OF WORKING PAPER: Progress of the role of the associated bacteria on the biology, rearing and behavior of Anastrepha obliqua, A. serpentina and A. striata

AUTHOR (S): M. Aceituno-Medina & E. Hernández

ORGANIZATION: Programa Moscafrut (SAGARPA-SENASICA

Abstract:

The objective of the project was to understand and harness the role that microorganisms play in the larval and adult biology of Anastrepha obliqua, A. ludens, A. serpentina and A. striata to improve the SIT component as part of area-wide integrated pest management. The contribution corresponds to the achievement of 7-experiments grouped in 3-goals that corresponded: 1) Develop methods of using beneficial bacteria to replace costly ingredients in larval diets in SIT applications. 2) Determine the effect of radiation on the symbiotic communities in target species. 3) Explore the use of symbionts as probiotics provided to adult sterile males before their release to significantly improve sterile male. The main outputs corresponded enriched larval diets for A. obliqua and A. striata that were developed and evaluated in the experiments, “Effect of feeding lactobacillus‐based probiotics on the gut microflora, growth survival and immune response of larvae of Anastrepha obliqua (Diptera: Tephritidae)”, “Effect of the addition of autogens bacteria in the larval diet on the biological traits of Anastrepha striata (Diptera:Tephritidae)” and “Presence of Fusarium spp. and incorporation of mycotoxin sequestering agents, enzymes and remedial microorganism in larval diets of Anastrepha obliqua”. The effect of the irradiation and the use of enriched diets with probiotics to diminish the effect were explained by the experiments “Midgut morphology in the irradiated males of Anastrepha ludens” and “Morphological and population bacteria changes in the gut of Anastrepha ludens during the metamorphosis”. Diets for adult in order to determine the use of symbionts to improve the sexual competitiveness was evaluated by the experiments, “Survival and competitiveness of males of Anastrepha obliqua and Anastrepha serpentina fed with enriched food with bacteria” and “Attractiveness and sexual competitiveness of A. obliqua males (Diptera: Tephritidae) fed with enriched food with Providencia rettgeri”.

Objective 1. Develop methods of using beneficial bacteria to replace costly ingredients in larval diets in SIT applications.

Experiment 1 Effect of feeding lactobacillus‐based probiotics on the gut microflora, growth survival and immune response of larvae of Anastrepha obliqua (Diptera: Tephritidae) Martha Nallely Guillén Rivera1, Roberto Rodríguez Bamaca2, Morfa Carrasco1, Marysol Aceituno-Medina3, & Emilio Hernández3 1Instituto Tecnológico de Tapachula, (ITT), 2Instituto de Biociencias (UNACH), 3Programa Moscafrut (SAGARPA-SENASICA),

The addition of probiotics to the post-irradiation diet improved sterile male performance in copulatory tests in the laboratory (Ben-Ami et al. 2010). Therefore, the bacteria enriched diet of mass-reared sterile males is a valid approach to improve their sexual performance.

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Gatesoupe (1999) defines probiotics as “live microbial cells administered to cultured organisms to colonize the digestive tract and improve their immune response”. Among the probiotic we can found the lactic acid bacteria, which stand out for their easy multiplication, production of antimicrobial compounds (bacteriocins, hydrogen peroxide, organic and lactic acids), and the stimulation of the non-specific immune response of the host (Gatesoupe, 2008). Studies have demonstrated the beneficial effect of the addition of such bacteria in the culture of insects like as Drosophila melanogaster (Sharon et al., 2011) y Aphis mellifera (Vázquez et al., 2012). Probiotics have been shown to have several modes of action, competitive exclusion of pathogenic bacteria through the production of inhibitory compounds, improvement of water quality, enhancement of immune response of host species and enhancement of nutrition of host species through the production of supplemental digestive enzymes (Gomez Gill et al., 2000). The beneficial effects that probiotics can provide insect larvae and adults is a promising alternative, however the failure or success of their addition is directly related to the characteristics of the artificial diet used as growth substrate. Based on this, the main objective of this work is to determine the immune response and nutritional effects of Lactobacillus rhamnosus on larvae and adults of Anastrepha obliqua. In this experiment was determined the effect of the inoculation of L. rhamnosus on artificial diet. Five milliliters of the lactic acid bacteria inoculum (1x109 CFU/mL) were sprayed per 100 g of artificial diet. Probiotic incorporated diet was incubated at room temperature (32°C). The inoculated artificial diet was maintained for 8 days to assess the survival of the bacteria in the diet. Every 24 h a sample was taken diet and inoculated in MRS agar. The control diet was sprayed with sterile culture medium. L. rhamnosus strain (BIENA, Quebec, Canada) were grown in 100 mL of de Man, Rogosa and Sharpe (MRS) broth (Difco, France). Specifically, 10 mL aliquots were used of the MRS broth which were inoculated with 10 mL of 24 h bacterial cultures and incubated under aerobics conditions at 37°C. Immediately, 1 mL of each culture were taken and serial dilutions were performed in 10 mL of saline solution for bacterial count, spreading 10 μL of each dilution in triplicate in MRS agar plates (Difco, France). Similarly, 100 μL aliquots of each culture were taken after 24 h and 48 h by performing serial dilutions as described before and plated them for bacterial counting to determine the bacterial colony count. The plates were incubated under aerobics conditions at 37°C for 48 h. The count was expressed in colony forming units per mL (log CFU/mL). The results showed that L. rhamnosus survived very well on MRS agar and artificial diet (Figure 1 and 2). Strain L. rhamnosus was able to maintain its viability for 8 days on artificial diets. After 6 days of diet storage at room temperature, we observed a concentration of 3.4 x 108 cfu/g during first 5 days, which means the L. rhamnosus was able to survive and reproduce. After 5 days the concentration was decreasing rapidly to reach 0.53 x 107 cfu/g. The supplemented diet looked in good condition, no molds proliferation neither bad odor were observed. The results obtained until now can describe the type of cell found in the hemolymph of males of A. obliqua feeding without bacteria. We conclude that good stability is obtained with L. rhamnosus on artificial diet. The further actions would be to study the biological effect and immune response of these probiotic in mass- reared fruit fly.

Experiment 2 Effect of the addition of autogens bacteria in the larval diet on the biological traits of Anastrepha striata (Diptera:Tephritidae) Ingrid Berenice Puon Ordoñez1, Marysol Aceituno Medina2, Carmen Ventura González2, Miguel Salvador Figueroa1, Emilio Hernandez2

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1Instituto de Biociencias, Universidad Autónoma de Chiapas, 2Programa Moscafrut (SAGARPA- SENASICA)

This work was to evaluate the effect of the addition of microorganisms on the larval diet on the biological traits of Anastrepha striata. The bacteria species that were isolated from the digestive tract of wild flies of A. striata corresponded to Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterobacter cloacae and Enterococcus faecalis. The effect of the preservatives, methyl paraben and sodium benzoate at different pH were performed on each species of bacteria to determine their effect on viability. The bacteria were inoculated in a started-diet using the 5x105 and 5x107 CFU/g. The results indicated that the addition of Enterobacter cloacae allows 33% larva-pupa survival, whereas Klebsiella pneumoniae inoculation without metabolites allows the increase the pupa weight from 13.56 to 19.27 mg, survival larva-pupa from 20.33 to 33%.

Experiment 3 Presence of Fusarium spp. and incorporation of mycotoxin sequestering agents, enzymes and remedial microorganism in larval diets of Anastrepha obliqua Marysol Aceituno-Medina, Carmen Ventura & Emilio Hernández Programa Moscafrut (SAGARPA-SENASICA)

The storage of corn cobs for production of the corn cob fractions takes place outdoors for several months, allowing the proliferation of fungi of the genus Fusarium, and thus the presence of mycotoxins in corn cob. Also, sometimes presents bacteria of the genus Morganella and Serratia, considered as harmful both for mass rearing of fruit flies. The aim of this study was to determine the presence of Fusarium spp and bacteria of the genus Morganella and Serratia. The effect of addition of some mycotoxins sequestering agents, enzymes and remedial microorganisms in diet was evaluated.

Presence of Fusarium spp. The molecular identification was made using the primers: Fg16NF/R F. graminearum, FP82F/R for F. oxysporum, FACF/R for F. acuminatum and J1Af for F. avenaceum.

Presence of entomopathogenic bacteria. The molecular of Morganella spp. and Serratia spp., was made using primer ERIC1 (5'-TGAATCCCCAGGAGCTTACAT-3') y ERIC2 (5'- AAGTAAGTGACTGGGGTGAGCG-3') and pyrosequencing. The results obtained for microbiological analysis indicate that although there is presence of Fusarium oxysporum, the larval performance was not affected. However the lowest larval yields were obtained with corn cob with the presence of Fusarium graminearum, Morganella morganii and Serratia marcescens. It is necessary to determine the lethal concentrations for Anastrepha flies since only in this study was performed to identify the microorganisms in the corn cob, but not to define the lethal concentration of the microorganism and / or of the toxin.

Remedial microorganism. The effect of the incorporation of mycotoxin sequestering agents, enzymes and remedial microorganisms in larval diets of Anastrepha obliqua was carried according a random design with six treatments (Table 2), for each treatment were carried 10 replicates. An experimental unit consisted in 6 trays with 6 kg of diet seeded with 1.7 ml of eggs. The mycotoxin sequestering agents and enzymes additives were added in powder to the mixture of the solids of the diet. In the case of the solution of remedial microorganisms, this was directly added to the water diet preparation. The variable response larval performance (larvae / g diet) and larva weight (mg). Proposals concentrations for this experiment were selected through bioassays previously conducted

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experimentally (trays with capacity of 1 kg of diet). With regard to information obtained by using different additives in order to observe any improvement in performance, statistical analysis found no significant difference with respect to the positive control in the number of larvae per gram of diet and larval weight. These results indicate that, when the corn cob fractions are contaminated with Fusarium mycotoxins and presence of Morganella spp and Serratia spp, is possible to decrease the risk using mycotoxin sequestering (Carvet, Aflaban, Xtract 6930) and recover the number of the larvae per gram, larval weight and not differences were observed in the mate capacity.

Objective 2. Determine the effect of radiation on the symbiotic communities in target species Experiment 4 Midgut morphology in the irradiated males of Anastrepha ludens A.Vázquez Gómez1, M. Aceituno-Medina2, M. Salvador1, J. Toledo3 & E. Hernández2 1Instituto de Biociencias, Universidad Autónoma de Chiapas, 1Programa Moscafrut (SAGARPA- SENASICA), 3El Colegio de la Frontera Sur

The irradiation damage on the cells of the midgut in adult males of Anastrepha ludens (Loew) was described. The pupae were irradiated at 60 and 80 Gray and the control that was not irradiated. The irradiated and control flies were dissected when the adult reach the age of 1and 7 day old. Fertile males of 1 and 7-days old showed and intestinal epithelium with degenerated possibly due to early aging and result of the intense activity by the cells. Irradiation caused cellular changes in the midgut of sterile males, the severity of these alterations in the cells depended on the dose, the age of the insect section and evaluated. Regarding the presence of bacteria in the midgut of these were found in lesser amounts on the first day old, fertile males had the gut bacterial load of irradiated males , meanwhile the intestines of male 7-days old contained bacteria mature established along midgut biofilms, bacteria are presented in smaller quantities in male fertile irradiated . Bacteria that formed bacterial biofilms were bacilli, cocci, coccobacilli and diplococci. The changes caused by irradiation were described and the images indicated that the midgut of male degenerates completely days after treatment with irradiation as in some species of insects, although bacteria found we cannot ensure the physiological state in which these are found, it could be that they are not fulfilling the functions have been attributed .

Experiment 5

Morphological and population bacteria changes in the gut of Anastrepha ludens during the metamorphosis G.R. Váquez-Martínez1, Marysol Aceituno-Medina2, Miguel Salvador1 and E. Hernández2 1Instituto de Biociencias, Universidad Autónoma de Chiapas, 2Programa Moscafrut (SAGARPA- SENASICA),

Bacteria play an important role during insect holometabolism. In fruit flies, bacteria of the family Enterobacteriaceae have been shown to improve the longevity and sexual performance of male breeding individuals, therefore, deficiency in sexual competitiveness has been associated with changes in the bacterial communities of the digestive tract. These changes are mainly caused by the dissimilarity between wild and wild-type environmental conditions, which include the absence of wild-type microbial sources acquired by horizontal transfer. However, metamorphosis could represent another factor that alters the bacterial communities of the digestive tract. For this reason, the development of the

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digestive tract during larva-pupa-adult transformation and its influence on the enterobacteria population was characterized using PCR-DGGE analysis. It was found that the intestine of the larva gives rise to that of the adult, the main morphological changes of the digestive tract were reported at 5, 9 and 16 days of pupation. During the transformation larva-pupa-adult, decreased the number of species of enterobacteria, but a group of species remained free of changes (conserved species).

Objective 3. Explore the use of symbionts as probiotics provided to adult sterile males before their release to significantly improve sterile male

Experiment 6 Survival and competitiveness of males of Anastrepha obliqua and Anastrepha serpentina fed with enriched food with bacteria I. Gómez Alonso1, M. Aceituno Medina2, J.P. Rivera-Ciprian2, M. Salvador1, J. Toledo3 & E. Hernández2 1Instituto de Biociencias, Universidad Autónoma de Chiapas, 1Programa Moscafrut (SAGARPA- SENASICA), 3El Colegio de la Frontera Sur

Consistently, the sterile males of A. obliqua and A. serpentina during the competitiveness test showed lower values than the wild males. Previous experiments indicate that flies increase their sexual competitiveness when consume enriched foods with bacteria during the sexual maturation period. In this experiment, bacteria of flies of the same species were isolated and subsequently the effect of foods containing such bacteria on biological attributes A. obliqua and A. serpentina were tested. The bacteria isolated from the midgut of A. obliqua were Providencia rettgeri, Serratia marcescens, Citrobacter freundii, Erwinia spp., Staphylococcus aureus, and Enterobacter cloacae. While, in A. serpentina the isolated species were Serratia marcescens, aglomerans Pantoea, Providencia rettgeri, Staphylococcus aureus, Staphylococcus xylosus and Chryseobacterium indologenes. These bacteria were added in Mb® food and sucrose concentration of 5x104 and 5x1010 UFC/ g. The results indicated that the survival under stress conditions of the males of A. obliqua increased from 3 to 7 days when fed with the mixture enriched with sucrose containing all the bacteria at a concentration of 5x104 UFC/g. A. serpentina did not showed any significant effect . The male sexual competitiveness of A. obliqua and A. serpentina increased in a 21.1 and 19% when P. rettgeri and P. aglomerans were added in Mb® food 21.1 and 19% , respectively, whereas in induction of the sterility not was observed any significant benefit in both species of fruit flies.

Experiment 7 Attractiveness and sexual competitiveness of A. obliqua males (Diptera: Tephritidae) fed with enriched food with Providencia rettgeri Linnet Roque1, Emilio Hernández2, Marysol Aceituno-Medina2, Carmen Ventura2, Jorge Toledo3 & Edi A. Malo3 1Instituto de Biociencias, Universidad Autónoma de Chiapas, 2Programa Moscafrut (SAGARPA- SENASICA), 3El Colegio de la Frontera Sur The West Indian fruit fly, Anastrepha obliqua, (Macquart) is the second most important tephritid fruit fly in Mexico by infest mango, plum and guava fruits. To control, the Mexican government implemented the use of the Sterile Insect Technique (SIT). The SIT involves the mass production and sterilization of flies. However A. obliqua laboratory males used in SIT are less selected by the wild females during the competitiveness tests. The objective of this work was compared the effects on males feeding with Mb® food for fruit flies (Mubarqui Aerial Services, Cd. Victoria, Tamaulipas, México) enriched with

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Providencia rettgeri diet against males feeding with Mb® food alone, on the males mating competitiveness, capture of females using traps baited with males fed with the enriched food and sex pheromone components. The results indicated that the males fed with food enriched with P. rettgeri increased the mating competitiveness and the captured females during the field cages tests. Seven volatile compounds were identified during the calling of A. obliqua males ((Z) -3-nonenol, nonadienol, sesquiterpene, (E,Z) -α-farnesene, β- farnesene (E,E) -α-farnesene and farnesene isomer. Results were discussed considering the use of bacteria in the mass rearing technique of A. obliqua adults to improve the sexual competitiveness of males from laboratory than wild males.

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TITLE OF WORKING PAPER: IDENTIFICATION OF THE DIFFERENT ENTEROBACTERIA PRESENT IN GUT OF DIFFERENT STAGES OF THE WILD AND MASS REARED MEDFLY INSECT

AUTHOR (S): Teresa Navarro, Jaime García de Oteyza

ORGANIZATION: TRAGSA, Bioplanta Caudete de las Fuentes, Valencia, Spain

SHORT SUMMARY OF PAPER Abstract:

The Mediterranean fruit fly, Ceratitis capitata (Wied.), is one of the most harmful agricultural pests worldwide causing sizable economic damages, as well as an archetypal model for implementing the Sterile Insect Technique (SIT). The Valencian Community has been applying the SIT during the last 10 years.

Key aspects for a successful implementation of this technique are, among others, quality parameters of the released insects such as mating competitiveness and longevity, and rearing efficiency. Mass reared insects show a drift on its gut microbiota when compared to the wild ones due to the rearing and irradiation processes. Latest works have demonstrated that manipulation of this microbiota in flies´gut could have a beneficial effect on their fitness, especially on mating competitiveness and longevity.

After isolation and identification of the different enterobacteria present in different stages of the wild and mass reared insect gut, potentially beneficial ones were selected.

The objective of this work was to recover these selected microorganisms in the mass reared insects in order to regain their valuable effects for the SIT programme.

In the previous work, two inoculation systems based on biofilm formation on the incubators airstones were tested but no differences on quality parameters were found. Taking occupational health and safety requirements as a prerequisite within rearing process, a new inoculation system has been tested and results will be discussed.

Finally, preliminary screening data for yeasts present in wild and mass reared larvae and adults medflies will be shown.

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TITLE OF WORKING PAPER: Wolbachia-based Strategies to Increase Sterile Ceratitis capitata Wiedemann (Diptera: Tephritidae) Quality in Support of SIT

AUTHOR (S): Sevgi Bakir1, George Tsiamis2, Fuat Aydin3, Kostas Bourtzis4, Secil Abay3, Osman Ibis5, Aydin S. Tuncbilek ORGANIZATION: Erciyes University, Faculty of Science, Department of Biology, Kayseri, Turkey SHORT SUMMARY OF PAPER Abstract:

Ceratitis capitata (Wiedemann), the Mediterranean fruit fly (medfly), is a very destructive pest of citrus and other crops in numerous nations all through subtropical and tropical locales. In spite of the fact that SIT is generally used to control C. capitata, the sterilizing irradiation methodology effects the bacterial community of the fly and the mass- rearing techniques characteristic to the SIT often lead to a lessening in the mating capacity of the released males. To fully assess the role of bacteria, it is essential to understand the diversity of microbiota harboured by irradiated and non-irradiated populations. The study of the medfly gut bacterial communities utilizing culture-dependent approaches’ revealed that adults gut communities were dominated by two phyla: Proteobacteria, and Firmicutes. We find that the Enterobacteriaceae seen most commonly bacteria families present in the gut of both irradiated and unirradiated adults. The irradiation procedure not clearly influences the gut bacterial community of the wild and laboratory medfly samples compared with their untreated control. On the other hand we revealed significant differences in some bacterial species between the wild and laboratory medfly. Enterobacteriaceae build up with five genera in laboratory but six and seven genera in wild medfly samples from Antalya and Mersin provinces, respectively. These are Klebsiella, Pantoea, Morganella, Enterobacter and Providencia in both irradiated and unirradiated adults in wild samples by the culture-dependent. One genus, Stenotrophomonas, was found only in laboratory medfly samples. Male and female adults gut community’s abundance in 1- and 5-day-old adults appeared higher than in their corresponding in 15-day-old adults. Totally eight genera and nine species were identified by the culture-dependent methods in medfly samples. Reasons for the bacterial variations might be related to the med ﬂies living condition or nourishment sources. We did not find Wolbachia and any other reproductive endosymbionts in the samples collected from Turkey.

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TITLE OF WORKING PAPER: Identification of bacteria with detrimental and beneficial effect on the larval survivorship of the Mexican fruit fly (Anastrepha ludens)

AUTHOR (S): Erin Schuenzel, Norman Barr, Bacilio Salas, Don Vacek, Hugh Conway

ORGANIZATION: University of Texas Rio Grande Valley, USDA-APHIS-PPQ

SHORT SUMMARY OF PAPER Abstract: The Mexican fruit fly, Anastrepha ludens (Loew) is a major pest that attacks the fruit groves in the southwest region of the United States and Mexico. Female Mexican fruit flies can lay up to 1500 eggs during their lifetime representing a major threat to citrus and other fruit industries in these regions. One method to control the population of Mexican fruit flies is through biocontrol, specifically sterile insect technique (SIT). Key to this method is raising millions of sterile males per week for release. Because of the sheer number of sterile males released each week, the reproductive success of female fruit flies is severely reduced. For SIT to be successful, sterile males must be released constantly and bacteria are now being considered as a resource to decrease the cost of SIT by increasing larval-adult survivorship of the sterile males. Bacteria can play a duel role in the success of a fruit fly colony. As a probiotic, bacteria have been found to increase adult survivorship and mating success. Conversely bacteria can also act as pathogens that, if introduced, can wipe out the larvae in a colony or severely reduce the number of larvae that pupate.

To assess the potential of a bacterium as a probiotic or pathogen, the identity of the bacterium must be known. Bacteria and fungi were isolated from the eggs and larvae of A. ludens as well as the diet and from surfaces in the mass-rearing facility. The universal primers were used to amplify and sequence the 16s rDNA region of bacteria and ITS region of fungi. To date, 660 isolated microorganisms were amplified, sequenced and identified. Fifty isolates were identified as fungi with Candida sp. and Zygosaccharomyces bailii with the most isolated. 560 isolates of bacteria were identified with Providencia sp. and Pseudomonas sp. being the most prolific. The remaining fifty isolates were not identifiable to a known genus. Note that isolation was biased towards microorganisms that are culturable and colonies chosen for identification similar to other pathogenic microbes.

Pathogenicity trials that included larval survivorship and pupal weight were performed on over 400 of the bacterial and fungal species. Five bacterial species in two genera (Morganella and Providencia) and one fungus (Zygosaccharomyces) were found to have pathogenic effect on A. ludens. One bacterial species (Pseudomonas) was also found to have possible inhibitory effect on Morganella. Further genotyping and pathogenicity testing revealed that all isolated strains of Morganella sp. are economically detrimental to the mass-rearing needed for SIT. Determination of the best method for detecting this bacterium is on-going with future comparison between selective media, 16s rDNA sequencing and real-time PCR based on the hdc gene.

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TITLE OF WORKING PAPER: A review of Wolbachia in tephritids with a pest control perspective

AUTHOR (S): Mariana Mateos, Humberto Martinez, Kostas Bourtzis, Antonios Augustinos George Tsiamis, Jorge Toledo, Brenda M. Moran Aceves, Silvia Lanzavecchia, Diego Segura, Karina Guillen

ORGANIZATION: Texas A&M University and others

SHORT SUMMARY OF PAPER Abstract: The taxonomic distribution, prevalence and phenotypic effects of Wolbachia in tephritids is reviewed. Approximately half of the ca. 100 tephritid species examined to date are reported to harbor Wolbachia (of supergroups A and B). The reported prevalence in natural populations is highly variable among species (and populations). The potential biases in detection and identification of Wolbachia, and approaches to ameliorate them, are discussed. In several species and populations, prevalence is close to 100%. However, occurrence of “transient” infections is common in some species. The main Wolbachia phenotypic effect reported in tephritids is Cytoplasmic Incompatibility, but there is a paucity of such studies, possibly due in part to the difficulty of manipulating Wolbachia infection (by artificial transfer and by antibiotic curing). In general, other common effects of Wolbachia, such as defense against RNA viruses, have not been evaluated. Considerations for the identification and evaluation of Wolbachia strains that could be used in population suppression programs on the basis of the “Incompatible Insect Technique” are discussed.

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