Working Material

IAEA-314-D42015-CR-.4 LIMITED DISTRIBUTION

WORKING MATERIAL

ENHANCING VECTOR REFRACTORINESS TO TRYPANOSOME INFECTION

FOURTH RESEARCH COORDINATION MEETING

ORGANIZED BY THE JOINT FAO/IAEA DIVISION OF NUCLEAR TECHNIQUES IN FOOD AND AGRICULTURE

November 27 – December 1, 2017 Tanga, Tanzania

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

Table of Contents

1. INTRODUCTION AND CURRENT STATUS ...... 5 2. REVISED LOGICAL FRAME WORK ...... 13 3. INDIVIDUAL WORK PLANS FOR THE NEXT 12 MONTHS ...... 21 4. Recommendations ...... 38 5. AGENDA ...... 39 6. LIST OF PARTICIPANTS ...... 47 7. ANNEX I: WORKING PAPERS ...... 51

1. INTRODUCTION AND CURRENT STATUS

Tsetse and African trypanosomosis. Tsetse flies (Diptera: Glossinidae) are the only cyclical vectors of African trypanosomes, which are the causative agents of human and animal African trypanosomiases (HAT and AAT, respectively). HAT is endemic to 36 countries in sub-Saharan Africa with about 70 million of the inhabitants at risk. In 2009, the number of new cases of HAT reported to WHO dropped below the symbolic number of 10,000 (WHO update Febr. 2016). However, given that the disease affects hard to reach rural populations, and that active surveillance in war-torn areas is non-existent, the disease prevalence numbers are undoubtedly a gross underestimation. The related disease AAT causes estimated losses to African agriculture of US$ 4.5 billion per year and has a profound effect on development of the continent.

The most economically important African trypanosomes are transmitted during the bite of the tsetse fly. The AAT-causing trypanosomatids T. vivax, T. congolense and T. brucei brucei are major pathogens of livestock. Humans are only infected by T. b. rhodesiense and T. b. gambiense.

Following the success of the SIT programme in Zanzibar and the PATTEC (The Pan African Tsetse and trypanosomiasis Eradication Campaign) initiative of the AU (African Union), i.e. reducing the fly population size, interest in the use of SIT for tsetse and trypanosomosis control is increasing. To date, the IAEA-supported SIT projects have been in areas without human sleeping sickness, but future projects could include areas of actual or potential human disease transmission. In such projects it would be ethically unacceptable to release flies capable of transmitting the parasite to humans. Therefore, methodologies to prevent parasite transmission by released sterile male flies must be developed. To achieve this goal, a complete understanding of the underlying mechanisms involved in vector competence is necessary. The current CRP proposal addresses this issue by proposing experiments that will decipher interactions between the tsetse fly, its symbiome, and pathogenic African trypanosomes.

Tsetse-trypanosome interactions. The transmission cycle starts when a tsetse fly feeds on an infected vertebrate host. Distinct trypanosome species exhibit different developmental cycles in various tissues and organs of the tsetse fly vector. T. vivax has the simplest life cycle, with development occurring exclusively in tsetse’s mouthparts. So far, only limited information is available on factors that affect T. vivax development in the fly. For T. congolense and T. brucei initial establishment of infection occurs in the fly midgut, with subsequent maturation in the fly’s proboscis and salivary gland, respectively. During this journey, these parasites undergo several rounds of differentiation and proliferation, finally ending in the metacyclic form that is end-stage infective to the vertebrate host. The success rate of trypanosome colonization in the tsetse fly is low and often fails during initial establishment of the fly’s midgut. Tsetse present a chitinous peritrophic matrix (PM), on which assembly an array of proteins, that lines the midgut. The PM serves as a formidable front-line barrier that trypanosomes must circumvent in order to establish an infection in the tsetse midgut. The flies also mount a robust innate immune response, and experimental down regulation of the Imd pathway increases midgut infection prevalence. Similarly, RNAi silencing of tsetse’s immune-responsive glutamine/proline-rich (EP) protein promotes trypanosome establishment, and reactive oxygen species are also important determinants of resistance to infection. Following colonization of tsetse’s midgut, trypanosomes again differentiate and then migrate to the fly’s salivary glands (SGs). This process, which represents another significant bottleneck that mediates parasite transmission, is not fully understood. The cardia (the tissue that forms the junction between tsetse’s foregut and midgut), which is an immune-active tissue, produces nitric oxide synthase and contains increased 6 levels of nitric oxide, reactive oxygen intermediates and hydrogen peroxide (H2O2). From the cardia, only a few trypanosomes will succeed in migrating to, and colonizing tsetse’s SGs. The tsetse fly SG and proboscis function as biotopes to which parasites adhere, multiply within and undergo their final re-programming into infectious metacyclic forms. Information on the molecular composition of saliva is scant, and was derived mainly from in silico interpretation of a SG EST library and a limited ‘proteomics’ analysis. Recent progress through this CRP action has provided more information on the key players on the pathogen host interactions in tsetse’s midgut and SGs.

Now additional information is emerging regarding the underlying mechanisms that facilitate 1) trypanosome adaptation to different tsetse microenvironments, and 2) trypanosome circumvention of tsetse immune responses during their developmental journey through the fly.

Insect Symbiosis. Symbiosis is ubiquitous in nature and has had significant consequences in promoting evolution and biodiversity. This is particularly true for insects that establish both endo and ectosymbioses. These symbiotic associations are just starting to be understood. Symbiotic microorganisms affect different aspects of their insect host’s physiology, including development, nutrition, reproduction, speciation, defense against natural enemies and host preference.

Currently, insect symbiotic associations can be divided into at least three categories. The first category includes mutualistic symbionts that provide their host with nutrients such as amino acids and vitamins. In return, the mutualist receives an nutrient-rich, protective niche in which to reside and thus can maintain a highly reduced gene inventory. The second category includes commensal symbionts that provide their hosts with the ability to survive heat stress and develop resistance to parasitic infections. Finally, parasitic symbionts manipulate the reproductive properties of their hosts, inducing phenomena such as parthenogenesis, feminization, male-killing and cytoplasmic incompatibility (CI).

Tsetse symbiosis. All tsetse flies, examined to date, harbor an obligate symbiont of the genus Wigglesworthia. This relationship is ancient (50-80 million years), and likely serves to complement tsetse’s vertebrate blood-specific diet. In fact, Wigglesworthia’s highly reduced genome encodes several vitamin biosynthesis pathways, the products of which are absent from vertebrate blood.

Two populations of Wigglesworthia exist in tsetse. The first is found within bacteriocytes, which collectively comprise an organ called the ‘bacteriome’ that is located immediately adjacent to tsetse’s midgut. This population of Wigglesworthia presumably supplements metabolites absent from its host’s diet. Tsetse’s second population of Wigglesworthia is found extracellularly in the female milk gland. This milk-associated population of cells colonizes developing intrauterine larvae. Genomics and metabolomics investigations identified crucial nutritional components provided by Wigglesworthia that support tsetse fecundity. Supplementation of tsetse’s blood diet with these crucial metabolite cocktails enabled the maintenance of flies that lack Wigglesworthia but still maintain fecundity. Such Wigglesworthia-free flies allowed for functional investigations into Wigglesworthia’s crucial role in tsetse physiology.

Recent experiments demonstrate that Wigglesworthia also has an immuno-modulatory role in tsetse. Adult tsetse that lack this symbiont (GmmWgm-) exhibit an immuno-compromised phenotype compared to their wild type counterparts (GmmWT). When challenged with trypanosomes, gut infections are established in a large percentage of GmmWgm- flies. Conversely, GmmWT flies are highly resistant and can efficiently clear parasite infections. This obligate symbiont must be present during immature larval stages in order for tsetse’s immune system to develop and function properly during adulthood. In addition to stimulating immune system development, Wigglesworthia also 7 indirectly enhances immunity in wild type individuals. This phenotype results from the decreased expression of an anti-trypanosomal protein encoding gene, pgrp-lb, in tsetse’s bacteriome organ. PGRP-LB functions to protect Wigglesworthia by scavenging Wigglesworthia-derived PGN, thus suppressing up-regulation of host immune system activation. Decreased expression of pgrp-lb in the absence of Wigglesworthia results in higher susceptibility to parasitism.

Tsetse’s second symbiont, Sodalis, is a commensal bacterium found in all lab-colonized tsetse lines and some natural populations. This polytropic bacterium is found intra- and extracellularly in tsetse’s gut, hemolymph, salivary glands and reproductive tract (Sodalis is also transmitted to intrauterine larvae via maternal milk secretions). Unlike Wigglesworthia, Sodalis from different tsetse species are closely related, thus indicating this bacterium’s recent association with its tsetse host. Sodalis exhibits genotypic traits similar to those found in several free-living microbes, and can be cultured outside of tsetse and is amenable to genetic manipulation. Furthermore, Sodalis co- inhabits tsetse’s gut along with pathogenic trypanosomes. Recent data suggest that tsetse’s midgut microbiota (Sodalis and Wigglesworthia) can modulate trypanosome development. These characteristics make Sodalis an ideal candidate for use in tsetse paratransgenic disease control strategies.

Tsetse’s third symbiont is the alpha-proteobacterium Wolbachia, making it the most successful symbiont on earth. This bacterium infects up to 40% of arthropods, including insects, terrestrial crustaceans, spiders, scorpions and springtails, as well as filarial nematodes. Wolbachia, which is strictly intracellular, is mainly transmitted maternally to progeny with the egg cytoplasm (although more and more reports emerge in literature showing that horizontal transfer events are more common than earlier anticipated). Wolbachia found in different arthropod species exhibit varying levels of co-evolution with their host. As such, this bacterium can present parasitic, commensal or mutualistic (obligatory) phenotypes. In order to enhance their own spread through host populations, Wolbachia cause a number of reproductive phenotypes in many arthropods, including CI, male killing, feminization and parthenogenesis. Furthermore, as part of their co-evolutionary physiological inter-relations at cellular and metabolic levels, Wolbachia can manipulate host fitness, fecundity, immunity and longevity, development and even sexual behaviour. As recently shown in hybrids between closely related Drosophila hosts, mutualistic Wolbachia can lose host-directed replication control, thus over-replicating so as to transform into pathogens. Such artificial hybrid- host systems can serve as sensitive biomonitors for studying symbiont dynamics in vivo.

As demonstrated recently, Wolbachia mediate more host-pathways than was earlier anticipated. For example, Wolbachia protects D. melanogaster from infections with RNA viruses, whereas similar studies on the Wolbachia-mediated protection against entomopathogenic fungi in D. melanogaster are still inconclusive. The analyses of Wolbachia-mediated protective effects against pathogenic microbes has recently been extended from the Drosophila host system to Aedes aegypti and Anopheles stephensi mosquitoes. In these cases, Wolbachia infection interferes with the establishment of viral infections, as well as infections with Plasmodium and filarial nematodes.

In addition, Wolbachia-infected Ae. aegypti have reduced lifespans, as expected from the phenotype in its original host. Wolbachia-infected Ae. aegypti are less likely to transmit viral diseases such as dengue (virus), which require specific incubation periods in their mosquito vectors before transmission to humans can occur. The application of this virulent Wolbachia is therefore a novel strategy for fighting vector-borne diseases, such as dengue, malaria and filariasis, by interfering with vector biology. As recently demonstrated, such artificially infected mosquitoes spread rapidly in the wild and provide dengue protection to their novel host. Because of these seminal discoveries, Wolbachia continues to attract an applied-research interest as a novel biocontrol agent for arthropod pests and vectors such as mosquito-transmitted malaria or even tsetse fly-transmitted trypanosomiasis. Many tsetse species and populations harbour closely related but distinct 8

Wolbachia strains at different titres and with different infection prevalences. In addition, a number of tsetse species have Wolbachia sequences inserted into their chromosomes. In the laboratory- reared G. m. morsitans, crossing studies between Wolbachia-infected and -uninfected individuals have demonstrated the expression of strong CI. Surveying the potential capacity of natural Wolbachia infections to functionally interfere with trypanosome transmission is of pivotal importance.

During the course of the CRP Spiroplasma was identified as a fourth bacterial symbiont, so far exclusively, in G. fuscipes fuscipes and G. tachinoides members of the palpalis subgroup. Spiroplasma is a genus of wall-less bacteria belonging to the class Mollicutes that is associated with diverse plants and arthropods. Spiroplasma is grouped into three major clades as has been shown by 16S rRNA gene-based analysis as well as multi locus sequence typing (MLST) studies. MLST analysis identified two strains of tsetse-associated Spiroplasma, present in G. f. fuscipes and G. tachinoides, both grouping in the citri clade.

Spiroplasma density in G. f. fuscipes larva guts is significantly higher than in guts from teneral and 15-day old male and female adults. In gonads of teneral and 15-day old insects, Spiroplasma density is higher in testes than ovaries, and is also significantly higher in live versus prematurely deceased females. This outcome indicates a potentially mutualistic association.

Symbiont-based tsetse control strategies CI-inducing Wolbachia strains can cause high levels of embryonic lethality. Thus, this symbiont can be applied for population suppression of insect pests and disease vectors in a way analogous to SIT (this process is referred to as incompatible insect technique, IIT). As recently shown, strong and promiscuous CI-inducing Wolbachia strains can be used as a novel, environment-friendly tool for the control of insect populations such as Ceratitis capitata (the Mediterranean fruit fly), Bactrocera oleae (the olive fly) and Culex pipiens and A. aegypti (mosquitoes). Wolbachia induced CI can also be used to augment the sterility caused by irradiation and enhance the efficacy of current SIT methods. Furthermore, Wolbachia-induced CI can be applied as a natural driver system for spreading transgenic insects and maternally transmitted modified symbionts (in paratransgenesis approaches, see below) through populations. Because transgenic organisms are likely to be less fit than their wild-type counterparts, transgenic traits must be actively driven into the population, in spite of fitness costs by means of population replacement. By a variety of mechanisms, Wolbachia- infected females have a reproductive advantage relative to uninfected females, allowing infection to spread rapidly through host populations to high frequency in spite of fitness costs. Hence, Wolbachia can be exploited to drive ‘valuable’ transgenes or modified symbionts into vector populations for disease control. Investigations into the reproductive biology of tsetse provide insight into mechanisms by which reproductive endosymbionts (Wolbachia or Spiroplasma) may manipulate host physiology. Transcriptomic and proteomic analyses have now identified tsetse products that are transmitted along with sperm to females from males in the presence and absence of Wolbachia, and provide the foundation for novel methods to interfere with reproduction.

Tsetse’s symbiont population is also amenable to the use of paratransgenesis as an approach to reduce fly susceptibility to infection with trypanosomes. Expression of parasite resistance genes in Sodalis, followed by the bacterium’s introduction into laboratory reared sterile tsetse prior to release in the field, can now be harnessed for disease control. Sodalis paratransgenesis, coupled with SIT, would further decrease the risk of trypanosomiasis transmission to the human populations in areas designated for control.

Viral pathogens An essential aspect of SIT is the ability to establish large colonies of tsetse lines. Several species of tsetse can be infected with the Glossina pallidipes salivary gland hypertrophy virus (GpSGHV). 9

Infection can cause salivary gland hypertrophy (SGH) and significantly reduces the fecundity of the infected flies. Although SGH syndrome prevalence is likely to be very low in wild tsetse populations (0.5-5%), in G. pallidipes mass rearing facilities, prevalence can reach up to 80% and cause colony collapse. For example, high prevalence of SGH led to the collapse of the G. pallidipes colony in Seibersdorf, Austria in 1978 and 2002. The first report of the SGH was described in wild populations of G. pallidipes in the 1970s, but the pathology was later observed in other tsetse fly species from several African countries. The causative agent of this syndrome was described initially as a rod-shaped, enveloped DNA virus averaging 70 by 640 nm in size. This virus was associated also with testicular degeneration and ovarian abnormalities, and its presence affected the development, survival, fertility and fecundity of naturally or experimentally infected flies. Mother- to-offspring transmission, either transovum or through infected milk glands, is thought to be the mode of virus transmission in natural tsetse populations. In colonized tsetse the main route of virus transmission is horizontal, this being facilitated by artificial silicon membranes that are used for large-scale fly feeding. SGH syndrome has been observed in two other dipterans, the narcissus bulb fly Merodon equestris and the housefly Musca domestica, but recently a SGHV look-alike was found in the parasitic wasp Diachasmimorpha longicuadata. It remains to be elucidated whether additional viruses are present in tsetse.

Although PCR detection of GpSGHV in old G. pallidipes shows widespread asymptomatic virus infection (up to 100%), only 5-10% of the infected individuals develop SGH symptoms. Why some infected flies show symptomatic infection whereas others remain asymptomatically infected remains unclear. Although a positive correlation was found between symptomatic SGH and increased virus copy number (which indicates an accumulation effect of the virus related to the SGH symptoms), other unknown factors related to the fly’s genetics and interaction with its microbiota (symbionts) cannot be excluded. Preliminary data indicate a possible negative correlation between virus infection and Wolbachia. Therefore, it will be important to analyze the interaction between virus infection and tsetse’s other associated microorganisms (Wolbachia, Sodalis, Wigglesworthia, Spiroplasma and trypanosomes) in different tsetse species. Currently, the genetic analysis of two genotypes of GpSGHV (Ethiopia and Uganda isolates) with differential pathologies has been completed. Out of the 174 open reading frames, at least 140 of these are transcriptionally active and 83 encode confirmed proteins based on SG analysis. Some potential target genes for intervention have been identified. The most divergent open readings frames were used to determine genetic diversity SGHV found within tsetse in sub-Saharan Africa.

Dissection of tsetse-microbial associations towards vector and trypanosomiasis control. Tsetse’s bacterial symbionts are important during all fly life stages. They may also impact trypanosome vectorial competence and hence could enhance the efficacy of SIT programs. We seek to resolve four key questions related to tsetse’s microbial community:

 Can the elucidation of tsetse-trypanosome molecular interactions assist in the development of novel methods and approaches to reduce or prevent the transmission of trypanosomes by irradiated tsetse flies?  Can tsetse symbionts be used to develop novel vector and disease control tools, complementary to the SIT?  Can the characterization of tsetse’s symbiome and viral pathogens improve the efficacy of SIT?  Are tsetse symbionts (including the gut microbiota), and the fly’s competence as a vector of trypanosomes, affected by radiation?

These questions are expanded in the following paragraphs.

Tsetse-trypanosome molecular interactions 10

The transmission of major medically and veterinary important trypanosome species (T. brucei ssp., T. congolense and T. vivax) relies on the specific biological relationship between the parasites and the blood feeding tsetse fly. Indeed, depending on the trypanosome species, the parasite has to go through an obligatory developmental cycle that varies from a short cycle in tsetse’s mouthparts (T. vivax) to a longer, more complex life cycle in the fly’s midgut and mouthparts (T. congolense) or midgut, mouthparts and SGs (T. brucei spp). For both T. congolense and T. brucei, the molecular interplay at different developmental stages in the fly will determine if the parasite develops to its final infective stage. The elucidation of these interactions is essential to understand the determinants of tsetse vector competence for a given trypanosome species population and how they can be affected. High resistance (refractoriness) to trypanosome infection has been demonstrated both in laboratory lines as well as in natural populations. Understanding the genetic basis of tsetse’s resistance (vector competence) will help to develop tools to enhance fly refractoriness to trypanosome infection.

Tsetse microbiota All laboratory colonized tsetse harbour three distinct, maternally-transmitted bacterial endosymbionts. Additionally, some colonized tsetse are also infected with Spiroplasma and SGHV. Field-captured flies are also colonized with a population of bacteria transiently acquired from their environment. In other vector arthropod systems, gut microbiota prevent establishment and/or transmission of pathogens. Thus, acquiring a better understanding of the symbionts and pathogens present in the tsetse vector, and the physiology that underlies their interactions, may allow us to manipulate tsetse flies so that they exhibit increased refractoriness to trypanosome infection.

Symbiotic organisms and novel control tools Incompatible insect technique, which is based on the mechanism of Wolbachia-induced CI, has been successfully tested for control of the agricultural pests, Ceratitis capitata (the Mediterranean fruit fly) and Bactrocera oleae (the olive fly), under laboratory conditions. Also, Wolbachia- induced CI has been successfully used as a driving mechanism of desirable traits in mosquito A. aegypti populations under laboratory and field conditions in Australia and Brazil. Such approaches should be considered, alone and/or in conjunction with SIT, for the control of tsetse flies and trypanosomiasis.

At present, SIT has been proven effective in reducing or eradicating isolated tsetse populations. This method relies on the massive release of sterile male flies, which could result in a temporary increase in the number of potential trypanosome vectors in the release zones. As such, the use of a tsetse that present a trypanosome-resistant phenotype would render SIT a much less controversial component of integrated vector control strategies.

Tsetse’s commensal bacterium, S. glossinidius, is ideally suited for use in paratransgenesis because: 1) it resides in different tsetse tissues (midgut, haemolymph, salivary glands) and in close proximity to the pathogenic trypanosomes; 2) it can be cultured and genetically modified in vitro and made suitable to delivery effector molecules; 3) recombinant Sodalis can be re-introduced into female flies; 4) re-introduced recombinant Sodalis are maternally transmitted to larval offspring via milk secretions; and 5) due to large-scale gene erosion, Sodalis is metabolically dependent on its tsetse host niche, suggesting that this bacterial symbiont cannot survive outside of the fly. A key component of Sodalis-based tsetse paratransgenesis is the identification of anti-trypanosomal effector molecules that be used in the paratransgenic system. The tsetse-trypanosome interaction research proposed in this CRP will provide several potential candidates as anti-trypanosome effector molecules to be delivered by Sodalis.

4. Effects of radiation on symbionts and pathogens 11

Before their release into the field for SIT, male tsetse are irradiated to render them sterile. This radiation treatment may alter the bacterial community of the fly so as to create an imbalance between the different bacterial communities in the gut and other organs/tissues. Hence, this could have an impact on the tsetse’s physiology and trypanosome vector competence. Understanding the effects of radiation may enable us to design responses that address them in a manner that optimizes SIT efficacy. In addition, radiation may also result in the development and isolation of mutant strains of endogenous symbiotic bacteria leading to novel insect symbiotic associations that may affect tsetse vector competence. Moreover, in the context of the possible application of a paratransgenic approach to enhance refractoriness to trypanosome infection in released tsetse flies, evaluation of the impact of radiation on the transformed bacterial community is essential.

In conclusion, the elucidation of the molecular interactions between the host, it’s symbionts, and associated pathogens can have profound effects on the development and application of efficient control strategies for tsetse flies and trypanosomiasis. The current CRP aims to characterize and develop methods to harness the tsetse fly-trypanosomes-symbiont tripartite association in order to: (a) unravel the molecular interplay among tsetse flies, symbionts, and trypanosomes, (b) characterize tsetse microbiota under field and laboratory conditions as well as in the presence / absence of tsetse pathogens and trypanosomes, (c) develop novel, symbiont-based and SIT- compatible control tools for tsetse flies and trypanosomiasis, and (d) determine the effects of radiation on tsetse symbionts and pathogens. We believe that this initiative will lead to better and more cost-effective SIT programmes aimed at reducing tsetse populations and trypanosomosis. A better understanding of the vector-trypanosomes-symbionts tripartite association is essential to develop methodologies that will lead to the enhancement of refractoriness of the tsetse flies to trypanosome infection.

5. Genomics and transcriptomic research The following genomics resources relevant to tsetse and its associated microbiota are publicly available: - VectorBase (www.vectorbase.org). The Glossina Genome Cluster project has completed the whole genome sequencing of G. pallidipes, G. austeni, G. brevipalpis, G. fuscipes fuscipes and G. palpalis gambiensis. Tissue-specific transcriptomes are also available to assist with genome assembly and annotation. - NCBI (http://www.ncbi.nlm.nih.gov/pubmed). The genetic analyses of the genomes of Ugandan and Ethiopian strains of GpSGHV and the MdSGHV are complete. This information is complemented with transcriptomic and proteomic data. Additionally, genomic resources relevant to tsetse’s symbiotic microbes are available at NCBI. - TriTrypDB (http://tritrypdb.org/tritrypdb/). A database containing comprehensive trypanosome- related genomics data. - Genomes Online Database (https://gold.jgi.doe.gov/). A World Wide Web resource for comprehensive access to information regarding genome and metagenome sequencing projects, and their associated metadata, around the world.

2. REVISED LOGICAL FRAME WORK

Project Design Verifiable Means of Important Assumptions Elements Indicators Verification Overall Objective: African countries continue to The objective of the N/A N/A suffer from tsetse fly‐vectored project is to elucidate human and animal African the molecular trypanosomoses (HAT and interactions between AAT, respectively). Prevention host‐symbionts‐ and treatment measures pathogens to improve remain ineffective. Drug SIT and SIT‐compatible resistance remains a problem. interventions for the The increasing demand for control of area‐wide integrated vector trypanosomosis by management approaches to enhancing vector control vector‐borne diseases, refractoriness to allow including where appropriate SIT to be expanded into the SIT as non‐polluting areas of potential suppression/eradication human disease component, and SIT will be transmission. expanded into areas of potential human disease transmission. Financial and human resources are available. Novel approaches will be accepted on ethical ground. Specific Objectives: 1. To decipher the 1. Mechanisms and 1. Reports 1. Tools and experimental molecular interplay molecules that and models for functional between vector‐ cause parasite publications. research are available. symbiont‐ resistance identified trypanosomes in G. morsitans morsitans and other tsetse species.

2. To characterize 2. Diversity and 2. Scientific 2. Biological (field and tsetse symbiome composition of reports and laboratory) material and next including gut tsetse symbiome peer generation sequencing and microflora. identified. reviewed bioinformatics tools are publications. available. 14

Project Design Verifiable Means of Important Assumptions Elements Indicators Verification

3. To determine the 3. Effects of 3. Scientific 3. Radiation services are effect of radiation on radiation on vector reports and available. vector symbiome. symbionts assessed. peer reviewed publications.

4. To develop 4. Symbiont‐based 4. Scientific 4. Symbionts can be innovative symbiont‐ approaches, in reports and manipulated as tools for the based strategies, in conjunction with peer control of African conjunction with SIT, to SIT, developed and reviewed trypanosomosis. control African validated. publications. trypanosomosis. Outcomes: 1. Molecular cross‐ 1. Factors and 1. Scientific 1. Tools and experimental talking between vector‐ mechanisms reports and models are adequate and symbiont‐ involved in peer valid. trypanosomes further molecular interplay reviewed characterized explored. publications.

2. Vector symbionts 2. Vector’s 2. Scientific 2. Available genomics and characterized and symbiotic partner(s) reports and bioinformatics tools are impact on host determined and peer optimal. physiology identified. impact explored. reviewed publications.

3. The impact of 3. Qualitative and 3. Scientific 3. Experimental tools are radiation on vectors quantitative reports and available. and symbionts. changes on the peer experimental reviewed systems examined. publications.

4. SIT used in areas with 4. Strategy for the 4. Projects 4. Technology adopted potential human use of SIT in areas implemented. sleeping sickness of potential human transmission through sleeping sickness the release of has progressed refractory tsetse flies significantly.

Project Design Verifiable Means of Important Assumptions Elements Indicators Verification Outputs: 1.a. Molecular interplay 1.a. Impact of 1.a. Reports 1.a. Molecular tools and of G. m. morsitans‐ pathogen and/or and peer experimental models pathogens‐symbionts bacterial symbiont reviewed available. characterized. infection in relation publications. to immunity, sex, behaviour, reproduction and fitness characterized.

1.b. Comparative 1.b. Immunity 1.b. Reports 1.b. Funding, molecular tools genomics and relevant genes and peer and experimental models transcriptomics of identified, and reviewed available. Glossina species and comparison with publications. outgroups performed other symbiotic to elucidate vector model systems competence. performed.

1.c. Factors that affect 1.c. Detected field 1.c. Reports 1.c. Molecular tools available. T. vivax infections in populations of T. and peer tsetse flies assessed. vivax in SIT‐target reviewed species. publications.

2a. Symbionts of 2a. Symbionts in 2.a. Reports 2.a. Biological material, trypanosome infected laboratory and field and peer genomics and bioinformatics and uninfected tsetse populations reviewed tools are available. species and hybrid identified and publications. colonies determined. characterized.

2b. Interaction(s) 2b. Reciprocal 2.b. Reports 2.b. Biological material, between trypanosome effects between the and peer genomics and bioinformatics and symbiome in model symbiome and reviewed tools are available. tsetse species and trypanosomes publications. hybrid colonies assessed. determined.

Project Design Verifiable Means of Important Assumptions Elements Indicators Verification 2c. Impact of viral 2c. Effect(s) of SGHV 2c. Reports 2c. Biological material, pathology on tsetse’s infections on and peer genomics and bioinformatics symbionts. tsetse’s bacterial reviewed tools are available. symbionts publications. characterized.

3a. The effect of 3a. Impact of 3a. Reports 3a. Irradiation damages hosts, radiation on tsetse irradiation in mass‐ and peer symbionts and pathogens in a vectors, their symbionts reared species reviewed dose and species‐specific and pathogens determined. publications. manner. determined.

3b. The mutagenic 3b. Effect in G. 3b. Reports 3b. Paratransgenesis effect of radiation on morsitans morsitans and developed. Sodalis. assessed. publications

4a. Wolbachia‐based 4a. CI in G. 4a. Reports 4a. Wolbachia is amenable to population suppression morsitans morsitans and characterization by classical and/or replacement characterized and publications genetic and molecular strategies assessed. Wolbachia impact approaches. on mating behaviour evaluated.

4b. Develop parasite 4b. Potential anti‐ 4b. Reports 4b. Symbiont‐based resistant paratransgenic trypanosome and peer trypanosome control tsetse. molecules identified reviewed strategies can be developed and successfully publications. against target vector species. expressed and their effect evaluated in paratransgenic tsetse. ACTIVITIES: 1. Submit CRP proposal. 1. Consultants 1. Report of 1. CRP proposal approved by meeting held Consultants IAEA committee. October 2011. Meeting and CRP proposal prepared.

Project Design Verifiable Means of Important Assumptions Elements Indicators Verification 2. Announce project 2. CRP announced, 2. Issued 2. Suitable proposals amongst established and research contracts and submitted and approved by vector entomologists, contracts and agreements. IAEA committee. trypanosome biologists, agreements virologists and submitted, symbiologists evaluated and forwarded to IAEA committee.

3. Organize first RCM to 3. 1st RCM held. 3. Working 3. Research activities started. plan, coordinate and material Reports published and review research printed and distributed following each activities distributed RCM. for 1st RCM.

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

5. Second RCM to 5. 2nd RCM held. 5. Working 5. Research activities analyse data and draft material continue, progress technical protocols as printed and satisfactory. required distributed for 2nd RCM; Research published in scientific literature and disseminated to member states and scientific community.

6. Continue R&D. 6. Research carried 6. Reports 6. Renewal requests and out by contract and and continued funding of RCM’s agreement holders. publications. and CRP.

Project Design Verifiable Means of Important Assumptions Elements Indicators Verification 7. Review the CRP after 7. Mid‐CRP review 7. Report of 7. Mid‐CRP review by Agency its third year. carried out. mid‐CRP committee is positive. review.

8. Convene third RCM 8. 3rd RCM held 8. Working 8. Research activities to evaluate and beginning of June 6‐ material continue, progress standardize protocols. 10, 2016 in Lyon, printed and satisfactory. France. distributed for 3rd RCM; Research published in scientific literature and disseminated to member states and scientific community.

9. Hold workshops on 9. Workshop held 9. Workshop 9. There is need for training; “Microbial May 30th to June 5, report. techniques, equipment and bioinformatic and in 2016 in Lyon, instructors are available. silico methods and France. Harmonized fluorescence procedures and microscopy.” trainees capable of implementing novel techniques

10. Continue R&D. 10. Research carried 10. Reports 10. Renewal requests and out by contract and and continued funding of RCM’s agreement holders. publications. and CRP.

11. Hold final RCM to 11. Final RCM held. 11. Final CRP 11. Research and review data and reach report. dissemination activities consensus. concluded.

12. Evaluate the CRP 12. CRP evaluation 12. CRP 12. CRP evaluation by Agency and submit evaluation carried out. evaluation committee is positive. report. report.

Project Design Verifiable Means of Important Assumptions Elements Indicators Verification 13. Summarize and 13. CRP members 13. 13. Manuscripts accepted and publish advances of CRP submit papers Publication in published. in a series of joint summarizing scientific publications. activities. literature.

3. INDIVIDUAL WORK PLANS FOR THE NEXT 12 MONTHS

Just Vlak Jan Abeele Peter Takac Imna Malele Serap Aksoy Serap Anne Geiger Soerge Kelm Astan Trqore Astan Trqore Drion Boucias Drion Barbara Mable Flobert Njokou Njokou Flobert Fathiya Khamis George Tsiamis Tsiamis George Ikbal Agah Ince Anna Malacrida Wolfgang Miller Abdelaziz Heddi Gisele Ouedraogo Gisele Ouedraogo Martin Kaltenpoth Martin Florence Wamwiri Activities / PI laboratories Mekonnen Solomon Mechanism of trypanosome resistance in model x x x x tsetse identified Comparative genomics of different Glossina x x x x x species and outgroups related to immunity Comparative transcriptomics of Glossinia in x x x response to trypanosomes x Role of symbionts in trypanosome transmission x x x x x x x x x x x x x Male accessory gland (MAG) function in x x x x reproduction (behavior) Role of symbionts in MAG function (mating x x x x behavior) Symbiome of infected and wild type (WT) lab x x x x x x x x x x x x x x x and field flies Symbiome parasite interaction in lab, WT and x x x x x x x hybrid lines

SGHV and microbiome interaction (pathology) x x x x x x x x SGHV functional genomics and diversity x x x x x studies

Effect of radiation on tsetse and partners x x x x x

Mutagenic effect on paratransgenesis x x

Wolbachia CI identification x x x x x x

Wolbachia impact on mating behavior x x x x Antitrypanosomal products x x x x x

Paratransgenic expression x x x

Symbiont transmission mechanism x x x x x x x x x Repository of reference materials x x Comply with export regulations (Nagoya) x x x x x x x x x x x x x x x x x x x x x

Tissue distribution, identification, prevalence and effects of radiation on symbionts in tsetse flies, Glossina spp. in Ethiopia Solomon M. Tessema, Andrew G. Parker and Adly M.M. Abd-Alla Abstract Tsetse flies (Glossina spp) vector pathogenic African trypanosomosis, which cause sleeping sickness in humans and nagana in animals. Additionally, tsetse harbour 3 maternally transmitted symbiotic bacteria that modulate their host’s physiology. Many organisms harbour symbiotic partners that contribute to metabolism or defence. These symbionts maybe transmitted vertically from parents to offspring or horizontally from host to host. The midgut microbiota of tsetse flies is of significant interest since it could be involved in the survival of host and the trypanosome. Our work focused on the tissue distribution, identification, prevalence and effects of radiation on symbionts in laboratory reared tsetse flies. The dissection of 50 Glossina pallidipes of both sexes indicated the existence of symbionts in the laboratory colony and the effects of radiation was investigated. These symbionts are required for the normal physiological activities. Keywords: Glossina pallidipes, radiation, symbionts, trypanosomosis

Planned for the next 12 months: 1. Screen tsetse flies for SGHV. 2. Screen G. pallidipes for symbionts. 3. Analyze the effect of radiation.

Gisele Ouedraogo Ecole National de l’Elevage et de la Santé Animale (ENESA) Laboratoire Vétérinaire Collaborators: Tsiamis, G, Demerbass, G, Traore A, Rayaisse J.B, Sidibe, I. and Seibersdorf Lab.

We evaluated the prevalence and coinfection dynamics between Wolbachia, trypanosomes, and SGHV in four tsetse species (Glossina palpalis gambiensis, G. tachinoides, G. morsitans submorsitans, and G. medicorum; n=3102) that were collected from 46 geographical locations in Burkina Faso, Mali, Ghana, Guinea, and Senegal (West Africa) between 2008 and 2015. Our study indicates a possible negative role of Wolbachia in SHGV and trypanosome transmission, thus providing novel insights that could be useful for the development and implementation of sterile insect technique-based population control strategies. During this study, we used PCR to identify the different types of trypanosomes, the presence of virus and Wolbachia in our samples. Work plan Planned for the next 12 months - We will identify the different strains of trypanosomes, virus and Wolbachia, after cloning and sequencing, to have an idea of different strains of the parasites or symbiont. - We will then design specific primers for new species or strains identified from the sequences we obtained after the first sequencing and cloning. - The final objective will be to develop a protocol for the detection of species identified by conventional PCR, PCR-RFLP or nested-PCR.

Institute of Tropical Medicine, Belgium Linda De Vooght, Jan Van Den Abbeele, Collaborators: Seibersdorf laboratory, P. Takac, W. Miller, A. Malacrida, A. Geiger, F. Njiokou, S. Kelm

During the last five years we have delivered the proof-of-concept that commensal Sodalis glossinidius can be genetically engineered to express and release functional anti-trypanosome nanobodies in in vitro culture conditions as well as in vivo in the tsetse fly, including the midgut where the parasite establishes an infection. Moreover, we successfully developed a Tn7-based methodology for an efficient chromosomal transgene insertion in Sodalis allowing stable and strong expression of different selected nanobodies that target the tsetse procyclic midgut trypanosomes. We succeeded in a sustainable colonization of the adult tsetse flies and their subsequent offspring with genetically modified Sodalis by means of microinjection of newborn third instar larvae. This implies that a tsetse fly colony can be established with flies harboring a transformed- Sodalis which is an essential requirement for the massive release of paratransgenic trypanosome-resistant tsetse flies in the context of SIT. We have demonstrated that, beside the maternal transmission through the milk gland secretion, the symbiont is also paternally transmitted to the offspring through the male-to-female delivery of a Sodalis-containing spermatophore. This paternal transfer could provide an additional route for

introducing genetically engineered symbionts into the natural tsetse fly population. In conclusion, we can state that the methodologies to establish a paratransgenic tsetse fly colony have been developed. However, the last but most difficult hurdle still to overcome to render paratransgenic tsetse flies refractory to trypanosome infection remains the identification of an expressible component that is active in the tsetse midgut with a high lytic potency against the procyclic stage, blocking parasite transmission by the tsetse fly. In addition, we have studied by a whole transcript RNA-seq analysis the impact of a trypanosome infection on the tsetse salivary gland tissue to try to understand how the fly tolerates this invasive infection. Moreover, we are studying how the Sodalis population is controlled in the tsetse fly and how it is impacting tsetse fly biology. For this, we have established a Sodalis-free G. morsitans morsitans colony.

Work plan for the last CRP-period and beyond: - To compare the vector competence of the stabilized hybrid colony between morsitans/centralis hybrid flies versus the two parental tsetse fly lines for T.brucei (collaboration: Seibersdorf lab; W. Miller); - To produce transformed Sodalis expressing new candidate anti-trypanosome proteins and evaluate for their anti-trypanosome activity in vitro and in paratransgenic tsetse (collaboration: A. Geiger; F. Njiokou, S. Kelm, A. Malacrida); - To evaluate the Sodalis-free status of the colony by specific Sodalis Stellaris-probes (collaboration: W. Miller) - To determine the impact of Sodalis on the tsetse fly survival, reproduction and vector competence for T. brucei & T. congolense.

Laboratory of Virology, Wageningen University, The Netherlands Just M. Vlak Collaborators: H. Kariithi, I. K. Meki, I.A. Ince, Seibersdorf laboratory

Abstract: For the last 4 years of the CRP research, we had proposed to address 5 major topics with the following major goals: (1) To identify the gene products (expressome) of salivary gland hypertrophy virus (SGHV) in the tsetse fly salivary glands; (2) To characterize the SGHV gene promoters responsible for the regulation and sequential expression of viral genes; (3) To identify the SGHV genes expressed when the virus is in the latent state as it occurs in the field and in ‘healthy’ tsetse fly laboratory colonies; (4) To identify the viral genes responsible for controlling the host defense responses; and (5) To determine the diversity of SGHV in wild tsetse populations in different African ecogeographic regions (compared to colonized flies). From the 5 topics, 4 original research papers and 1 review article have already been published in peer-reviewed journals. A further 4 original research and 1 review article are in revised submission stage, while 1 original research article is in preparation stage (scheduled to be submitted by the end of 2017 and early 2018). A PhD thesis is also being prepared for defense in 2018.

Work plan for the next period (until the end of the CRP) - Investigating the nature and possible role of the small RNA viruses in tsetse. - Investigating the origin and role of the GpSGHV-like sequences in G. m. morsitans and other Glossina species - Finishing four publications intended for the special issue of BMC Microbiology (January 2018), one paper to PLoS One journal, and another paper tentatively targeted for Journal of Virology Journal. - Finishing the thesis of I. Meki (submission due 2018) - Finish topic 2 of the proposed research.

ICIPE, Kenya Fathiya Khamis

Metarhizium anisopliae infection reduces Trypanosoma congolense multiplication in Glossina fuscipes fuscipes and its ability to acquire or transmit the pathogen Collaborators: J. Van den Abbeele, N.K. Maniania, R. St Leger, A. Zaidman-Remy, F. Wamwiri, S. Mekonnen, G. Tsiamis and S. Kelm Tsetse fly-borne trypanosomosis remains a significant problem in Africa despite years of interventions and research. The need for new strategies to control and possibly eliminate trypanosomosis cannot be over- emphasized. Entomopathogenic fungi (EPF) infect their hosts through the cuticle and proliferate within the body of the host causing death in about 3-14 days depending on the concentration. During the infection process, EPF can reduce blood feeding abilities in hematophagous arthropods such as mosquitoes, tsetse and ticks, which may subsequently impact on the development and transmission of the parasites. The objectives of the project are therefore to understand the tritrophic interactions between fungus/parasite/tsetse.

Achievements of the project:  Successfully developed method to obtain high infection rates of T. congolense in G. f. fuscipes;  Exposure of the flies, G. f. fuscipes to M. anisopliae GZP-1 and ICIPE 30 isolates resulted into 100% and 86% mortality after four days, while mycosis was 14% and 22%, respectively. The parasite load decreased from 8.7 x 107 at day 2 to between 8.3 x 104 and 1.3 x 105 T. congolense ml-1 at day 3 post-infection in both fungus-treated groups of flies. These results were confirmed by quantitative PCR  Effects of the entomopathogenic fungi in both transmission and acquisition of T. congolense was established during the course of the project. T. congolense-infected flies exposed to both fungal strains (WT 30 and GZP-1), could not transmit the parasite to mice while fungus-free Trypanosoma- infected flies continued to transmit the parasite to naïve mice. Also, entomopathogenic fungi infected flies fed on T. congolense-infected mice were not able to acquire parasite at day 4 post- exposure while flies in the control treatment continued to acquire T. congolense up to day 5.  There was no binding observed between the recombinant trans-sialidase proteins to GZP-1 in vitro. There was a significant drop (P < 0.0181, KW = 8.625) in total haemocyte count in both groups of fungus-treated flies as compared to the controls with further variation in individual haemocyte types.  Mycelial growth network within the insect host was traceable by scanning electron microscopy. Germinated spores could be observed at nearly all parts of the surface of the insect body from day 2 with no preference site. Extensive mycelial network was observed in the abdominal tissues especially at the midgut where the parasites are expected to be in abundance in case of infection. Micrographs from fungus-free flies clearly showed the midgut structures but no fungal hyphal bodies. Cross sections of day 5 samples showed possible point of interaction between the fungus and the parasite as the intricate fungal mycelial network was seen on and within the haemocoel.  Demonstrated that the infection of G. f. fuscipes by the entomopathogenic fungus negatively affects the multiplication of the parasite in the fly and reduces the vectorial capacity to acquire or transmit the parasite. Workplan for the next 12 months of the project: Collaborators: N.K. Maniania, A. Zaidman-Rémy and Aziz Heddi (i) The infection of adult G. fuscipes by M. anisoplaie has an effect on their hemocytes. Heddi’s Lab has expertise in insect hemocytes, which will be used to better understand their description and cytology. (ii) Effects of fungal metabolites on trypanasomes. (iii) Investigate whether infection by M. anisopliae has an effect on midgut and proboscis microbiome from G. fuscipes. (iv) Screen for anti-fungal products that delay fungal killing or screen for isolates that have low pathogenicity for compatibility with SIT.

University of Glasgow, UK Barbara Mable

Collaborators: I. Malele, Seibersdorf laboratory, F. Njiokou

Over the duration of the CRP, our focus has been on characterizing sources of variation explaining the association between tsetse species, trypanosomes and the endosymbiont Sodalis glossinidius in field- collected samples. We thus optimized and tested markers appropriate for identification of tsetse species, symbionts and blood meals obtained from vertebrates, as well as developing a panel of microsatellite markers for assessing population genetic structure in T. congolense Savannah. We also conducted a microsatellite-based population genetics analysis to identify the original source of the IAEA G. pallidipes colony maintained at icipe and to quantify levels of inbreeding.

In Nigeria, T. vivax and T. congolense Savannah were the main species of trypanosomes found in the three species of tsetse screened (G. palpalis palpalis, G. tachinoides, G. morsitans submositan) but there was variation in their prevalence among tsetse species and other trypanosomes were also found in lower numbers (Isaac, Ciosi et al. 2016 Parasites & Vectors, 9: 301). The nature of the host communities (i.e. in protected conservation areas vs agricultural regions) strongly influenced the diversity of trypanosomes detected, but there was very low prevalence of the endosymbiont S. glossinidius and none were amplified from G. tachinoides. In Kenya, we conducted a similar survey but also considered interactions among factors that could affect the relationship between tsetse and trypanosomes, including the presence of endosymbionts (Channumsin et al. 2018 BMC Microbiology special issue) and the host communities on which the flies fed (Wongserepipatana 2016 PhD Thesis, University of Glasgow). We compared two geographic regions with different habitats: 1) green forest in the Shimba Hills, where there are pastoral communities sharing the landscape with wildlife; and 2) savannah forest in Nguruman where there is less human activity. Geographic region had the strongest influence on the presence of trypanosomes but this was confounded by differences in age class distribution and gender biases between regions in the different species of tsetse found (G. pallidipes, G. austeni, G. longipennis and G. brevipalpis). Trypanosome prevalence was particularly high in the Shimba Hills and T. vivax and T. congolense Savannah were the dominant trypanosome species, although T. brucei was present at a substantial frequency. No association was found between T. vivax presence and presence or absence of Sodalis but both the endosymbiont and trypanosomes (of any species) were much rarer in Nguruman and the Shimba hills so there was not much power to test the association across regions. Blood meal analysis revealed that there was also a difference in the feeding behavior of flies in the two regions: in Nguruman most flies were found to have fed only once and buffalo and elephants were the primary hosts; in Shimba Hills a much larger number of flies fed multiple times and there was more evidence for feeding on humans and livestock, as well as wildlife. These differences could affect risk of exposure to trypanosomes and presence of endosymbionts. A major limitation of these studies is distinguishing infection from transient parasites based on PCR-based assays so these correlations are indicative but do not directly inform factors that affect refractoriness of tsetse to parasites.

The development of multiplex microsatellite PCRs to genotype T. congolense Savannah was completed. It is of little use in the field because the number of trypanosomes in field samples is too often too low to be able to amplify the microsatellites. However, it is very useful for characterization of T. congolense Savannah once they have been expanded into a vertebrate host in the laboratory.

The population genetic analyses of the IAEA colony revealed that it was not a hybrid population, as had been suspected, but likely originated from the Kenyan/Uganda border and that a genetic bottleneck was associated with the foundation and establishment of the colony. Inbreeding could thus be a concern for the genetic health of colonies to be used in SIT programmes.

Work plan for the next 12 months: We will develop a proof of concept sequence capture based platform (combined with Illumina deep sequencing) for detecting unculturable or low titre bacteria, initially using a test panel of human bacterial pathogens. We will test both whole-genome tiling approaches for high-resolution genotyping of particular focal pathogens and the use of existing multi-locus sequence based typing markers from multiple bacteria as probes for detecting and quantifying the microbial communities present in mixed samples. Both of these approaches could be applied to allow more reliable detection of endosymbionts of tsetse.

Vector& Vector Borne Diseases Research Institute (VVBDRI) – Tanga, Tanzania Imna Malele

Collaborator: F. Wamwiri, S. Kelm, A. Geiger, A. Malacrida, J. Maniania, P. Takac and Seibersdorf laboratory.

At VVBD the following were conducted in relation to enhancing tsetse fly refractoriness to trypanosome infection for the past 18 months. G. pallidipes from Kabuku, Doma (non HAT areas) and Serengeti were collected for analysis of gut microbiota. Wigglesworthia glossinidia and secondary endosymbiont were only isolated from G. pallidipes sampled from the coastal area (Kabuku). The secondary endosymbiont has been implicated to influence trypanosome establishment in tsetse. Apart from the dominance of the obligate endosymbiont Wigglesworthia glossinidia and the commensal endosymbiont Sodalis glossinidus there were rich diversity of bacteria. The diversity of gut bacteria were obtained by culture-dependent approaches and after enrichment under aerobic and anaerobic conditions, 16S rDNA of each bacterial isolate was PCR amplified and sequenced. The overall majority of bacteria identified belonged in descending order to the Firmicutes (64%), uncultured bacteria (16%), Actinobacteria (9%), Proteobacteria (7%), unclassified bacteria (3%), and finally Bacteroidetes (1%). Diversity of Firmicutes was found higher when enrichments and isolation were performed under anaerobic conditions than aerobic ones. Experiments conducted in the absence of oxygen (anaerobiosis) led to the isolation of bacteria pertaining to six phyla (48% Firmicutes, 22% Actinobacteria, 17% uncultured bacteria, 6% unclassified bacteria, 5% Proteobacteria and 2% Bacteroidetes), whereas those conducted in the presence of oxygen (aerobiosis) led to the isolation of bacteria affiliated to three phyla only (76% Firmicutes, 16% uncultured bacteria and 8% Proteobacteria). Phylogenetic analyses placed these isolates into 11 genera namely Bacillus, Acinetobacter, Mesorhizobium, Paracoccus, Microbacterium, Micrococcus, Arthrobacter, Corynobacterium, Curtobacterium, Vagococcus,and Dietzia spp.which are known to be either facultative anaerobes, aerobes, or even microaerobes. Here, we report the isolation under anaerobic and aerobic conditions of bacteria pertaining to phylogenetic clades comprising previously non-cultivated bacteria. The study shows that G. pallidipes fly gut is an environmental reservoir for a vast number of bacterial species, and could be important for the ecological performance of the fly and possibly on differing vectorial competence and refractoriness against African trypanosomosis. We also trapped flies from Serengeti and sequenced their gut microbiota; however, the detailed information is yet to be extracted.

The manuscript on bacterial diversity in the gut of G. pallidipes has been submitted to BMC Microbiology and part of the results were presented as a poster during the Area-Wide Management of Insect Pests 22–26 May 2017, Vienna, Austria

The plan to investigate on the use of entomopathogenic fungi (Metarhizium anisopliae) isolate ICIPE 30) as a tool for savannah tsetse and trypanosomes control at another other than Meatu was not successful as the fungi expired and the process to import a new batch could not be processed in time, hence the study was not continued.

The plan for the next 12 months: 1) To extract the information from the sequenced midgets obtained from G. m. moristans and G. pallidipes from Morogoro and Serengeti area and associate the occurrences of symbionts, trypanosome infection and various bacteria found in midgut and their role in tsetse refractoriness. 2) Finish the investigation of the interaction of SGHV with other gut microbiota from Glossina pallidipes and G. m. morsitans and any other sympatric tsetse species found in the coastal and Serengeti area 3) Publish the results.

KALRO - Biotechnology Research Institute Florence Wamwiri Collaborators: J.Maniania, S.Mekonnen, and the Seibersdorf Laboratory

Tsetse ﬂies (Diptera: Glossinidae), the primary cyclical vectors of pathogenic African trypanosomes, harbour a variety of maternally-inherited microbes including Wigglesworthia, Wolbachia, Sodalis and the salivary

gland hypertrophy virus (SGHV). Sufficient knowledge about tsetse-microbe interactions may contribute towards identification of novel strategies for vector and disease control. However, successful application of such strategies requires up-to-date baseline information on infection frequencies in the target populations. In summary, our contribution to the current CRP (a) We determined the infection frequency (Sodalis, Wolbachia) and concurrent trypanosome infection in 5 tsetse species (G. brevipalpis, G. pallidipes, G. f. fuscipes, G. austeni, and G. longipennis) from 5 populations in Kenya viz: Arabuko, Shimba Hills, Nguruman, Suba and Busia. Among the species the infection prevalence as follows: Wolbachia: G. austeni>G. brevipalpis>G. longipennis>G. pallidipes=G. fuscipes whereby infection in G. austeni was 100% and in G. pallidipes this was absent. Sodalis prevalence varied from 83% in G. brevipalpis, and decreased thereafter in G. pallidipes>G. austeni. Sodalis was absent from G. longipennis and G. fuscipes. Inter-population differences were also evident. (b) We characterised Wolbachia isolated from Kenyan G. austeni populations by Multi-Locus Sequence Typing (MLST), confirming limited strain diversity among the two populations sampled. (c) We also carried out infection frequency (Sodalis, Wolbachia) and concurrent trypanosome infection in G. m. centralis from 2 populations in western Zambia. In the remaining period of the CRP, we propose the following: (i) G. m. centralis: increase sample size and inclusion of and inclusion of females in analysis. Also to confirm re-test the ITS1-positive DNA samples with specific primers, and if possible to sequence the products obtained (ii) Investigate the presence of Spiroplasma in G. fuscipes field isolates

Department of Medical Microbiology, School of Medicine, Acibadem University, Istanbul, Turkey İkbal Agah İNCE

Collaborators: IK Meki, D. Boucias, S. Aksoy, and Seibersdorf Laboratories

The identification of expressed genes of viral entities is the main issue in recent genomic era. Expressomics, an integrated approach applying transcriptome and proteome outputs for data interpretation for Glossina hytrosavirus (GpSGHV) pathobiology, will be applied. Determination of the 3-prime untranslated regions (UTRs) has been performed (proof of concept) following to this progress exploring the conserved mechanisms related to viral pathogenesis will be undertaken. In addition, an integrated approach using transcriptome and proteome of infected cells performed to further knowledge on interactions between different tsetse isolates and the GpSGHV isolates are comparatively analysing currently by generation of genome and proteome maps. Knockdown of host protein factors related to viral infection will be investigated. As there is no available genetic recombination system for host as well as for virus (e.g. bacmid), in vitro dsRNA production will be used for generating knockdowns of target genes for clarifying the transcriptional regulation of this new group of viruses (Hytrosavirus). Production of genetic recombination systems as bacmids, will greatly enhance progress of this research proposal. The ultimate aim is to unravel the key virus-host factors responsible for viral infection and latency. This research will serve for the establishment of the effective mitigation strategies for tsetse rearing factories.

Major achievements in the first 18 months of the CRP-4 and next plan

 To unravel the evidence that Glossina hytrosavirus (SGHV) involves RNAi response, the total RNA and small RNA sequencing virus-infected and uninfected in tsetse flies (Glossina pallipides). o The final mapping of small RNA is achieved. The next is to determine the targeting hot spots of host response to viral infection.  Mapping of 3’ untranslated region of Glossina hytrosavirus transcript has been performed to contribute fine mapping of viral transcriptome and understand the termination motifs of viral transcripts. Termination motifs are important for development antivirals.

o The system of amplification of 3’ untranslated region was tested and approved. The next is to design size and melting temperature optimized gene specific primers to perform genome wide screening of UTRs

Centre for Biomolecular Interactions, Faculty for Biology and Chemistry, University of Bremen: Soerge Kelm Collaborators: J. Van Den Abbeele, A. Malacrida, F. Khamis, A. Geiger, I. Malele, G. Tsiamis, Seibersdorf laboratory and all groups sampling flies and groups analysing symbiomes The present research aims to acquire a better understanding of the role of trans-sialylation in the transmission and colonisation of trypanosomes between the insect vector and its mammalian host. Within the CRP the focus will be on further evaluating the potential of targeting sialidases expressed in the symbiome to prevent and/or eradicate trypanosomal colonisation and maturation in tsetse flies.

The following achievements have been made during the past 18 months: - Field tsetse fly samples were collected Nigeria, Ethiopia and Cameroon. - Phylogenetic relationship of tsetse flies collected has been determined. - Tsetse gut samples have been analysed for sialidase and sialic acid degradation activities using the enzyme assays and analytical methods established in the first periods of this CRP. Locations with higher density of sialidase positive tsetse guts have been identified to be resampled for bacterial screenings. - Samples from Nigeria, Ethiopia and Cameroon for virus and symbiont diversity studies within the CRP have been collected and shipped to the laboratory of Wolfgang Miller and to Seibersdorf. - Blood samples have been collected from cattle in Cameroon and Nigeria. The samples from Cameroon were analysed for the presence of trypanosomes and anti-TS antibodies. - A study on the effect of sialidase in tsetse gut on trypanosome colonisation has been started and initial preliminary results have been obtained. - Trans-sialidases from T. congolense has been further characterised to elucidate the role of the lectin domain for enzymatic - Screening of inhibitors of trans-sialidases has been initiated. First candidate molecules have been identified. - The potential binding of trans-sialidase (TS) lectin domains to entomopathogenic fungi was investigated. No evidence for direct binding of TS lectin domain to the fungi was obtained.

The following activities are planned for the next 12 months: - Further field tsetse fly midgut samples to be collected in Nigeria, Cameroon, Chad and Ghana as well as isolated bacterial cultures will be analysed for sialidase and sialic acid degradation activities. - Further samples to be collected in Nigeria, Cameroon, Chad and Ghana will be provided for virus and symbiont diversity studies within the CRP. - Existing bacterial isolates from tsetse midguts will be screened for sialidase activity - Nanobody libraries against T. brucei and T. congolense will be screened for anti-TS nanobodies - Attempts will be undertaken to collect and cultivate tsetse gut bacteria expressing sialidase from field samples - Existing metagenomic data from tsetse microbiomes will be screened for potential sialidase genes.

Seibersdorf, IAEA: Abd-Alla, A. Collaborators: W. Miller, A. Heddi, L. De Vooght, M. Kaltenpoth, G. Tsiamis, S. Mekonnen, J.Vlak, A.Ince, H. Kariithi

The Seibersdorf laboratory will be involved with several of the proposed work plans by providing services to CRP partners. Following the preliminary results suggesting a negative impact of irradiation treatment on tsetse symbionts, we plan to conduct a detailed study on the impact of irradiation on different aspects including the following:

Plan for the next 12 months:  Analyse the impact of irradiation treatment on the refractoriness of tsetse flies harbouring modified Sodalis (L. De Vooght and J. Van Den Abbeele)

 Analyse the impact of irradiation treatment on CHC (M. Kaltenpoth/T. Engl)  Analyse the impact of irradiation treatment on tsetse symbiont using FISH (A. Heddi)  Analyse the impact of irradiation treatment on tsetse symbionts in tsetse hybrids using FISH (W. Miller and A. Heddi)  Analyse the vectorial competence in tsetse hybrids (W. Miller and J. Van den Abbeele)  Analyse the impact of irradiation treatment on tsetse gut microbiota (G. Tsiamis)  Analyse the impact of SGHV on tsetse immune system (J. Vlak, H. Kariithi, and A. Ince)  Analyse the impact of SGHV infection on antimicrobial peptides (A. Heddi)  Analyse the impact of SGHV male reproductive biology (A. Malacrida)

Institut National des Sciences Appliquées de Lyon (INSA-Lyon), Villeurbanne, France Abdelaziz Heddi, Séverine Balmand, Florent Masson, Agnès Vallier & Anna Zaidman-Rémy Collaborators: S. Aksoy, B. Weiss, G. Attardo, A. Vigneron, G. Tsiamis, W. Miller, D. Schneider M. Kaltenpoth, T. Engl, A. Abd-Alla, I. Meki, Seibersdorf laboratory

Tsetse flies harbour three symbiotic bacteria, namely Wigglesworthia, Wolbachia and Sodalis. The modes of symbiont transmission, and their location in the insect tissues, were investigated during the last CRP program (Balmand et al., 2013, JIP). However, several features remain unresolved. Specifically, how these three symbiotic bacteria load and distribute during host development and across host tissues, and how they are perceived and controlled by the host immune system, remains questionable, although S. Aksoy team has made an impressive advance on this field. The achievements reached during the last 18 months are summarized as following: 1. We have organized in June 2016 a one-week workshop at INSA de Lyon, during which time ten students and researchers have experienced on two model systems (tsetse fly and cereal weevils) how to design FISH probes and how to conduct slide hybridization and cell imaging. We have analysed embryonic and post-embryonic stages and have monitored the distribution of the tsetse three endosymbionts as well as the primary endosymbiont of weevils. 2. We finally have performed a comparative immunity between Sitophilus weevils and Glossina tsetse. The genome of S. oryzae was assembled and annotated, and is now under the process of publication. We have used the weevil as a model system to identify key elements of the immune response that ensure the maintenance and control of endosymbionts, by using transcriptomic data and functional analysis tools that are available in this model. We also have tested the involvement of the tsetse fly orthologous genes in symbiosis regulation, including the pgrp-lb. 3. Through collaboration with the team of W. Miller, we have applied and obtained an Amadeus program for PhD student exchange. F. Masson, a PhD student in my team, has spent a two-week period in W. Miller Lab in Vienna to work on tsetse hybrids, and D. Schneider, PhD student from W. Miller team, has visited my Lab to perform FISH analysis on tsetse hybrids. 4. In collaboration with A. Abd-Alla team, we have analysed the expression of genes encoding antimicrobial peptides in the tsetse salivary glands infected or not with the virus GpSGHV.

University of Patras, Agrinio, Greece Eva Dionyssopoulou and George Tsiamis Collaborators: P. Takac, S. Aksoy, Seibersdorf laboratory Achievements In the beginning the profiling of laboratory tsetse populations of G. fuscipes fuscipes, G. morsitans morsitans, and G. pallidipes was analysed using 16S rRNA gene amplicon sequencing. The most abundant taxa, were Wigglesworthia (the primary endosymbiont), Sodalis and Wolbachia as previously characterized. Spiroplasma was identified as a new symbiont exclusively in G. f. fuscipes and G. tachinoides, members of the palpalis sub-group. Multi locus sequencing typing (MLST) analysis identified two strains of tsetse- associated Spiroplasma, present in G. f. fuscipes and G. tachinoides. Spiroplasma density was also examined by qPCR and in situ hybridization. The bacterial profile of three Glossina palpalis gambiensis laboratory colonies (Gpg-Burkina, Gpg-Intro and Gpg-Senegal) was also examined using 16S rRNA gene amplicon sequencing to evaluate the dynamics of the bacterial diversity within each G. p. gambiensis population and among them and to examine the relation

between the bacterial profile and the flies performance. The three G. p. gambiensis laboratory colonies displayed similar bacterial diversity indices and OTU distribution. Larval guts displayed a higher diversity when compared with the gastrointestinal tract of adults while no statistical significant differences were observed between testes and ovaries. Wigglesworthia and Sodalis were the most dominant taxa with the gastrointestinal tract in adults to be more enriched by Wigglesworthia while Sodalis were prominent in gonads. Interestingly, in larval guts a balanced co-existence between Wigglesworthia and Sodalis was observed. Sequences assigned to Wolbachia, Propionibacterium, and Providencia were also detected but to a much lesser degree. Clustering analysis indicated that the bacterial profile in G. p. gambiensis exhibits tissue tropism, hence distinguishing the gut bacterial profile from that present in reproductive organs.

Finally, in the frame of this CRP a workshop was organized during the 3rd RCM meeting entitled “The Characterization of Symbionts of tsetse flies via Bioinformatic Approaches”. Topics such as custom blast searches and amplicon data analyses were covered using a hands-on experience.

Future work: Complete the annotation of the Spiroplasma genome sequenced from G. f. fuscipes and perform comparative genomics

IRD, UMR 177 InterTryp « Interactions hôtes-vecteurs-parasites-environnement dans les maladies tropicales négligées dues aux trypanosomatidée », France Anne Geiger Collaboration: F. Njiokou, B. Ollivier (Aix Marseille Université, CNRS/INSU, Université de Toulon, IRD, Mediterranean Institute of Oceanography UM 110, Marseille), Seibersdorf lab.

Tsetse flies transmit African trypanosomes responsible for sleeping sickness in humans and Nagana in animals. This disease, fatal when untreated, affects many people with considerable impact on public health and economy in sub-Saharan Africa. The available drugs are inefficient and have even induced trypanosome resistance. Therefore, the investigations for novel strategies must continue, among them are alternative vector-based strategies such as the engineering of insects capable of blocking the transmission of the parasite. Field-captured flies are colonized with symbionts and a taxonomically complex microbiome acquired from their environment. In other insects, such as Anopheles sp., the gut microbiome was shown to prevent establishment and/or transmission of pathogens. In the light of this IAEA-CRP, our future plan is to complete and bring more knowledge regarding the interactions, between G. palpalis, trypanosomes and bacteriome, going on in African Trypanosomiasis foci, Fontem, Bipindi and Campo in Cameroon, by enlarging the number of field samples as well as from insectary flies. We will look at adult as well as teneral tsetse flies bacteriome. Then possible associations between the presence of a bacteriome and tsetse infection by trypanosomes will be researched. In case of positive associations, bacteria of interest will be isolated and then been given to feed insectary flies. These flies will then be challenged by trypanosomes to verify their actions on vector competence. Practically, this project will first involve entomological survey to enlarge our field sampling, in endemic area of Trypanosomiasis in Cameroon, with specific PCR identification of trypanosomes as well as bacteriome analyses using NGS. Statistics will be done using R package to identify associations between trypanosomes- bacteriomes. Bacteria isolation will be done using Hungate tubes. Experimental will then be performed.

Planning: Phase 1: larger entomological surveys and tsetse fly sampling to reinforce statistics of associations Phase 2: identification of the Glossina and trypanosomes species; Phase 3: characterization of the bacteriome by NGS; Phase 4: use of R package software for analysing the associations “trypanosomes-bacteria” presence. Phase 5: isolation of the bacteria of interest using adequate medium and oxygenation Phase 6: experimental infections using field isolated bacteria and trypanosome

Institute of Zoology, Slovak Academy of Sciences Peter Takáč

Collaborators: J. Van den Abbeele, S. Aksoy, G. Tsiamis, A. Malacrida, M. Kaltenpoth and Seibersdorf laboratory

Several contract and agreement holders had a requirement for live tsetse flies for experimental work, totaling 10,000 pupae of four species per year. The Institute of Zoology, Slovak Academy of Sciences, holds colonies of four tsetse species (G. morsitans morsitans; G. pallidipes; G. fuscipes fuscipes; G. palpalis gambiensis). The tsetse mass rearing facility provided supplies of requested pupae to all workplaces throughout the project duration. At the same time, will continue to supply the tsetse material with the aforementioned workplaces. During the project we investigated maternal transmission of symbionts to the progeny in cooperation with the other members of CRP as well as transmission of Peptidoglycan Recognition Protein (PGRP-LB). Flies that acquire fewer symbionts, and consequently less PGRP-LB, during juvenile development have immune defects and are at a disadvantage as young adults. We studied the molecular aspects of host-symbiont dialogue during colonization and establishment in host tissues, knowledge critical for paratransgenic applications. Bacterial transcriptome of females' milk glands, bacteriomes and larval guts were compared in all developmental stages to reveal bacterial interchange and intake during the larval development. At the same time we continued to study the impact of different kinds of diet on the gut symbiotic flora and on the fitness of the flies, related also to trypanosomiasis. In addition to their role in nutrition, there has been indirect evidence suggesting that the presence of symbionts might also enhance the establishment of trypanosome infections in the midgut and in this consequence the symbiont-trypanosome interactions. The impact of different kinds of meals on the gut symbiotic flora and the fitness of the flies were compared. As the already described endosymbionts produce metabolites to compensate the nutritional deficits in the host’s haematophagous diet and appears to be partially associated with the metabolism of B-complex vitamins essential for tsetse survival, the effect of yeast based meals and different B-complex vitamins’ meals (thiamine, pantothenic acid, pyridoxine, folic acid and biotin) on fitness and fecundity were analyzed. Finally, we studied the possibility to improving existing and developing new non-pesticide-based methods applicable in biological control of tsetse flies. One of the aims was to develop molecular genetic method to reduce the fecundity of tsetse flies. We therefore determined the gene expression in ovaries and uteri from both virgin and highly pregnant Glossina morsitans morsitans flies. Comparison of expression profiles between different stages of larval maturation allowed us to identify several candidate genes, which can be targeted by RNA interference (RNAi) in an effort to reduce tsetse fecundity.

The future plan for the next 12 months: Our investigations continue in transcriptome sequencies of different developmental stages of G.m. morsitans larvae and will be obtained genes related to specific stages of larval development . Silencing of selected genes related to larval development will be performed by RNA interference. The siRNA will be produced by dicer sirna kits and the effect of RNA interference will be observed on gene expression and fecundity. Fecundity will be monitored on flies injected by siRNA. Immunohistochemical experiments will be used to prove the expression of the genes in the selected tissues.

Department of Biology and Biotechnology, University of Pavia, Pavia, Italy Anna Malacrida Collaborators: S. Aksoy, G. Attardo, A. Heddi, P. Takac, L. De Vooght, J. Van Den Abbeele, Seibersdorf laboratory

Our research activities focus on : 1. The analysis of male reproductive physiology in Glossina m morsitans and on the impact of symbionts. - We investigated the impact of Wolbachia, Sodalis, Wigglesworthia on the molecular machinery that regulates the reproductive physiology of male tsetse. We derived RNA-seq data from the male reproductive tracts of wild-type and symbiont-free (aposymbiotic) flies. We found that the absence of symbionts a) depresses the expression profile of accessory gland genes, including those that may be associated with immunity and the production of seminal fluid proteins, and b) activates the expression of testis-specific genes with putative transport and reception functions. Metabolomics analyses are in progress.

2. Comparative genome analyses of six Glossina species; G m. morsitans, G pallidipes, G austeni, G fuscipes, G palpalis, and G. brevipalpis. - As a subgroup of the ‘Comparative Genome Analysis of 6 Glossina species’ annotation network, we a) performed a phylo-genomics analysis that allowed us to calibrate molecular clocks for the divergence time of these species, b) used the male reproductive genes we identified in G. m. morsitans to identify orthologous sequences in the other Glossina species, and infer their evolutionary patterns, and c) we identified several genes under lineage specific selective pressure which that be used to develop taxonomic tools. 3. Investigating the evolution of the molecular machinery that controls male reproductive physiology across Glossina species. - By analyzing the evolution of orthologous male reproductive genes in six Glossina, species we identified several genes that are under divergent selection in the different lineages. These genes provide sequences to be used to develop taxonomic tools. Moreover, we will investigate their functional role in the different tsetse species

Plan for the next 12 months: We will determine the functional role of male reproductive genes in relation to male fertility. The expression of these genes is affected by the absence of the symbionts.

Max Planck Institute for Chemical Ecology, Research Group Insect Symbiosis, Jena, Germany M. Kaltenpoth and T. Engl Collaborators: S. Aksoy, B. Weiss, V. Michalkova, D. Schneider, G. Uzel, P. Takacs, A. Abd-Alla, W. Miller, S. Balmand, A. Heddi

So far, we have characterized the cuticular hydrocarbon (CHC) profiles and mate choice decisions of Glossina m. morsitans upon perturbation of the microbial community by antibiotic treatment (tetracycline and ampicillin). While tetracycline treatment eliminates Wigglesworthia, Sodalis, and Wolbachia, ampicillin only affects Wigglesworthia, but not Sodalis and Wolbachia. Our results reveal significant differences in overall CHC profiles and in relative amounts of female contact sex pheromone between wildtype and antibiotic-treated flies, as well as reduced mating success of treated males and females (collaboration with S. Aksoy, B. Weiss, V. Michalkova, P. Takac). Gamma-irradiation on the other hand does disturb the titer of Sodalis and Wolbachia, but not Wigglesworthia. We could not detect any changes in CHC profiles of irradiated flies (collaboration with G. Uzel, A. Abd-Alla), excluding Sodalis and Wolbachia and pinpointing Wigglesworthia as the most likely candidate of the three endosymbiont as the cause of CHC disturbance in the antibiotic treated flies. However, due to the biology of all involved organisms we couldn’t distinguish between a direct impact of antibiotic treatment on host physiology and indirect effects via the symbionts. We were also not able to attribute changing in mating success of antibiotic treated flies exclusively to the CHCs, as dummy mating assays (using either dead flies whose CHCs were removed or Teflon dummies) could not be established. While the analysis of the gamma-irradiated tsetse flies (G. m. morsitans) did not reveal any effect of radiation dose, we found general handling effects of the mock radiation procedure, warranting care in general fly rearing, as this could have important implications for mate choice and sexual performance for SIT released flies in the field. In addition, we analysed the chemical profiles of G. m. morsitans and G. m. centralis parental and stable hybrid lines (collaboration with Wolfgang Miller and Daniela Schneider). First generation hybrid lines exhibited reduced total CHC amounts and especially reduced proportion of the female sex pheromone besides significant changes in the overall CHC profile. Stable hybrid lines however showed a reverse toward the parental Gmm CHC profiles and amounts. CHC analyses of SGHV infected G. m. morsitans flies are currently in progress and will be concluded until end of the year (collaboration with A. Abd-Alla, Seibersdorf) while we could not perform any analysis of purely Sodalis free or trypanosome infected flies, no corresponding samples were provided so far (collaboration with Jan Van Den Abeele). A workshop on fluorescence microscopic techniques to visualize symbionts and parasites in tsetse flies has been co-organized in Lyon, France (collaborators S. Balmand, A. Heddi).

Njiokou F.1, Kame Ngasse G.1, Nana-Djeunga H. 1, Melachio-Tanekou T.1 & Geiger A.2 1 Faculty of Science, University of Yaoundé I, Yaoundé, Cameroon 2 UMR 177, IRD-CIRAD, CIRAD TA A-17/G, Campus International de Baillarguet, 34398 Montpellier Cedex 5 Sodalis glossinidius and Wolbachia sp genetic diversities: relation with tsetse fly trypanosome infection in the Faro-Deo AAT in Cameroon Previous studies in Cameroon have shown that Sodalis glossinidius favours trypanosome establishment in Glossina palpalis palpalis in HAT foci. In order to verify if these observations are confirmed in an animal trypanosomiasis focus, investigations were extended to the Adamaoua region where the interaction between S. glossinidius, Wolbachia sp and trypanosomes hosted by tsetse flies were studied. Entomological prospections were done in four villages of the animal trypanosomiasis focus of Faro-Deo (Mayo-Dagoum, Golde Bourle, Tchabal Mbalbo and Mayo Riga). Tsetse flies were trapped using pyramidal traps. Species and teneral status were identified prior to dissection and collection of midguts. In the laboratory, Tsetse fly species were confirmed using ITS-1 (Dyer et al., 2008) while symbiont and trypanosome were identified by classical PCRs. Genotyping of Sodalis glossinidius samples were performed using four microsatellites loci genomic DNA (gDNA) 12/13, gDNA 15/16 and gDNA 21/22 gDNA 2/5 (Farikou et al., 2011) and Wolbachia samples, using MLST genes (gatB, cox A, hgpA, fbpA and ftsZ) (Baldo et al., 2006). Samples were subsequently prepared and sent for sequencing. For the two first villages (Golde-Bourle and Mayo-Dagoum), of the 315 tsetse flies dissected, 270 (85.72%) belonged to Glossina tachinoides species and 45 (14.28%) flies to G. m. submorsitans subspecies. For the 270 G. tachinoides, the parasite infection rates were 11.1%, 13.7%, 2.6% respectively for T. brucei s.l., T. congolense savannah type and T. congolense forest type. Symbiont infection rates were 37.63% and 68.1% respectively for S. glossinidius and Wolbachia sp. Statistical tests revealed no association between the presence of one or both symbionts and the level of infection of G. tachinoides by one single or more than one trypanosome species. For the two new villages 291 tsetse flies were trapped belonging to four species, dominated by Glossina tachinoides (91%). A total of 100 flies was analysed, 60 from Tchabal Babbo and 40 from Mayo Rig. Sodalis glossinidius prevalences were 63.33% and 52.5% while Wolbachia sp prevalences were 76.66% and 72.5% respectively in Tchabal Babbo and Mayo Riga. Trypanosome infection rates were 28.33% and 15% for T. congolense savannah type, 1.7% and 5% for T. congolense forest type, 1.7% and 2.5% for T. brucei brucei respectively in Tchabal Babbo and Mayo Riga. No association test was significant. For the four villages, no symbiont/trypanosome association test was significant. The genetic diversities were low with 1 to 2 amplicon band sizes per locus. The results of sequencing are expected before allele confirmation, haplotype identification and performing of symbiont haplotypes/trypanosome infections association tests.

In the next 12 months we will: - Analysing of sequencing - Blasting for species identification - Association tests haplotypes/trypanosome transmission - Analysing a new sample collected in 2017

Yale School of Public Health S. Aksoy/B. Weiss Collaborators: A. Malacrida, M. Kaltenpoth/T. Engl, G. Tsiamis, J. Van Den Abbeele, I. A. Ince, F. Wamwiri, I. Malele, P. Takac, A. Heddi and the Seibersdorf laboratory

Our research focuses broadly on the interactions between tsetse and its associated microorganisms. These microorganisms include parasitic African trypanosomes and tsetse’s indigenous bacterial symbionts. With respect to the goals of this CRP, our work focused on 1) characterizing the population of bacteria housed in the gut of field-captured flies, 2) performing comparative genomic analyses between tsetse species, 3) deciphering how tsetse’s bacterial symbionts mediate development and function of their host’s immune

system and reproductive biology, and 4) physiological mechanisms that underlie parasite transmission through tsetse. More specifically: 1. We used next-generation sequencing techniques to characterize the microbiota from distinct tsetse species captured at different geographic location. Additionally, we compared the enteric microbiotas of trypanosome infected versus uninfected individuals. Although we found no statistically significant difference in the taxonomic composition of environmentally acquired bacteria in the gut of infected versus uninfected flies, we did find that infected individuals housed significantly more Sodalis than did their uninfected counterparts. Results from these analyses are included in our manuscript submitted for publication the CRP special issue of BMC Microbiology. 2. We were involved with the comparative genomics of tsetse species project, which involved sequencing and annotating the genomes of five distinct tsetse. We focused specifically on the comparative analysis of reproductive and immune system related genes. 3. We investigated the functional relationship between tsetse’s indigenous bacterial symbionts and development of their host’s immune system. We discovered that tsetse must undergo their entire larval development in the presence of these microbes in for adults to present a functional cellular immune system. Additionally, symbiont-free tsetse lacks a structurally robust peritrophic matrix (PM). The PM lines tsetse’s gut, and provides a formidable physical barrier that trypanosomes must cross in order to establish an infection in tsetse’s midgut. In the absence of a robust PM, tsetse are highly susceptible to infection by trypanosomes. 4. We discovered that trypanosomes facilitate their passage through the tsetse vector by compromising the structural integrity of the fly’s PM. This process occurs when bloodstream form trypanosomes shed their variant surface glycoprotein (VSG) coat following entrance into tsetse’s gut. Liberated VSG molecules are taken up by tsetse’s cardia (which produces the PM), where they down-regulated expression of a microRNA (miR275) that controls production of PM-associated proteins. This results in the production of a weakened PM, which insect-adapted trypanosomes can more easily circumvent. 5. We collaborated with several CRP participants on other projects. These projects include: a) characterizing the association between tsetse’s bacterial symbionts and function of the fly’s reproductive system (A. Malacrida), b) characterizing the association between tsetse’s bacterial symbionts and the fly’s mating behaviour (M. Kaltenpoth/T. Engl), c) dynamics of symbiont colonization in larval tsetse (A. Heddi).

In the next 12 months (completion of the CRP) and beyond we will: 1. Further annotate the genomes from the five sequenced tsetse genomes, and submit the manuscript for publication. 2. Characterize Spiroplasma infection dynamics in G. fuscipes collected in Uganda. 3. Further characterize the functional association between tsetse’s indigenous bacterial symbionts and fly immune system development. 4. Further characterize the functional association between tsetse’s indigenous bacterial symbionts and fly reproductive biology.

Medical University, Vienna, Austria Wolfgang J. Miller Collaborators: M. Kaltenpoth, A. Heddi, B. Weiss, S. Aksoy and the Seibersdorf laboratory

In accordance with the definition of the biological species concept by Dobzhansky and Mayr, F1 hybrids between different Glossina species suffer from high embryonic lethality, plus complete hybrid male sterility. Such hybrid sterility has drawn attention earlier when artificially produced hybrids were considered for application strategies in order to control and suppress tsetse populations in the field. Moreover, mass-release of sterile hybrids would also allow for using significantly lower gamma-irradiation dosages to sterilize wrongly sexed females than currently applied high dosages for standard SIT ensuring complete sterilization of colony-bred males. Recent studies, however, strongly suggest that such rigorous irradiation treatments of male insects for standard SIT not only damage host spermatogenesis as desired, but also harm dividing somatic cells of gut epithelia, organelles, gut microbiota, as well as their native endosymbionts. Therefore, the potential mass-generation of naturally sterile Glossina hybrid males would improve significantly the fitness of released males in nature to fight vector-borne diseases such as Trypanosomiasis.

As recently demonstrated, symbiotic microbes can affect insect host fitness and fecundity, pathogen protection as well as their mating competence and success. Hence, rigorous sterilization of tsetse males with high dosages of gamma-irradiation could also destabilize or even demolish the complex symbiotic interactions between tsetse host and the native symbionts Wigglesworthia, Sodalis and Wolbachia. The tempting concept of applying hybrid-males that express innate sterility, however, was hindered by the fact that the potential large-scale generation of F1 hybrids for mass production and release is extremely laborious and hence not feasible. Only the establishment of stable hybrid colonies, i.e., the creation of a species nova tsetse fly, being incompatible per se with native Glossina species at their release site would circumvent this important biological and technical issue for future, successful pest control management. Such artificially generated host hybrid backgrounds, however, might have a massive impact on native symbiont loads. As recently reported by our group interspecies hybrids of neotropical Drosophila species dramatically increase titers of their native, mutualistic Wolbachia. Furthermore, we demonstrated that loss of symbiont titer control in hybrids triggers interspecies incompatibilities, such as high embryonic F1 mortality and complete male sterility. Upon Wolbachia knockdown in parents before mating, however, hybrid sterility can be partially rescued giving rise to fertile F1. Most similar to this system, we recently found that in the Glossina morsitans species complex, interspecies hybrids also exhibit increased Wolbachia loads compared to their corresponding non-hybrid parents. In addition, we have uncovered accompanying mild titer alterations in Sodalis and Wigglesworthia upon hybrid formation. However, not only general symbiont load, but also spatial distribution of symbionts might be altered in artificial hybrid backgrounds. As demonstrated in weevils, loss of host control over native symbiont localization has detrimental effects as it results in spreading of the primary symbiont in previously uninfected host tissues. We have performed detailed quantitative and high sensitivity in situ analysis of the spatial distribution of Glossina symbionts in artificial interspecies hybrid backgrounds, which is of pivotal interest for any further application in pest control management.

Specific achievements obtained over the last 18 month:

1. Generate antibodies against Wolbachia, Sodalis, Wigglesworthia, Spiroplasma and SGHV, testing and optimizing detection protocols (BW, SA). 2. Develop of 3 independent PCR and FISH protocols for solid low-titer symbiont detection (AH). 3. GmmWT female x GmcWT male cross results in wGmc-triggered strong cytoplasmic incompatibility (CI) and complete hybrid male sterility (Seibersdorf). 4. To reduce CI and partially restore hybrid male sterility, we generated GmcTET males via dosage- dependent tetracycline-feeding (3x versus 6x) (Seibersdorf). 5. GmmWT female x GmcTET male crosses resulted in overall higher pupae production plus emergence rate and partially restored F1 hybrid male fertility(Seibersdorf). 6. A stabilized Gmm x Gmc hybrid colony was established from 6x-treated Gmc fathers by consequent sib mating, currently at generation F25 (Seibersdorf). 7. Testing the hybrid colony for de novo postmating isolation: Backcrossing the stabilized Gmm/Gmc hybrid colony to both parental strains showed compatibility in both directions at F8 (bridging); however, when tested at F15 and F20 the hybrid colony was compatible with Gmm, but no longer with Gmc parents (Seibersdorf). 8. Testing the hybrid colony for de novo premating Isolation: Applying mate choice experiments with F1 of GmmWT x GmcWT and stabilized hybrids at F8/9 demonstrated that all hybrids are accepted randomly by both parental females and vice versa (absence of mate avoidance against hybrids – random mating), (Seibersdorf).. 9. Monitoring CHC pheromone profiles: Hybrids do not show any significant changes in their pheromone profiles and quantities compared to any of their original parents (MK), corroborating observed random mating. 10. Symbiont titer dynamics in hybrids versus parents: Only Wolbachia significantly overreplicate in F1 hybrids of GmmWT x GmcWT but not the other symbionts Sodalis and Wigglesworthia (AH). 11. Upon paternal symbiont knockdown (GmmWT x GmcTet), however, overreplication of Wolbachia is significantly reduced in F1 hybrids compared to F1 of GmmWT x GmcWT (Seibersdorf). 12. Symbiont transmission modes: F1 interspecies hybrids plus stabilized hybrid lines show clear signals of massive paternal leakage of mitochondria and Wolbachia.

Specific Aims planned for the next 12 months and beyond this CRP:

1. In-depth genetic (nuclear, mitochondria and symbionts) plus phenotypic analyses of the stabilized hybrid colony (Adly Abd Alla, Andrew Parker, Irene Meki, Daniela Schneider) 2. Testing hybrid colonies on vector competence (Jan Van Den Abbeele) 3. Providing high-sensitivity symbiont Stellaris probes for monitoring Sodalis dynamics and transmission modes upon paratransgenesis (Jan Van Den Abbeele)

General issue (All)

International exchange of biological materials is currently subjected to the rules and regulations of the Nagoya protocol. The Nagoya Protocol on Access to Genetic Resources and the Fair and Equitable Sharing of Benefits Arising from their Utilization to the Convention on Biological Diversity is a 2010 supplementary agreement to the 1992 Convention on Biological Diversity (CBD). Exchange of biological material should be clearly documented (source, owner).

4. Recommendations

1- Optimize protocols to detect and (semi) quantify components of tsetse’s symbiome using newly generated antibodies. 2- Studies on the effects of irradiation on tsetse’s microbiome in host dynamics should be completed. 3- Identify novel effector molecules for use in tsetse paratransgenic (Sodalis) control strategies. 4- In conjunction with SIT, explore the development of novel, emerging strategies for improving control of tsetse-transmitted trypanosomosis. 5- Adhere to the rules and regulations of exchange of biological materials and property rights (Nagoya protocol)

5. AGENDA

FOURTH RESEARCH CO-ORDINATION Meeting

JOINT FAO/IAEA DIVISION OF NUCLEAR TECHNIQUES IN FOOD AND AGRICULTURE

“Enhancing Vector Refractoriness to Trypanosome Infection”

TANGA, TANZANIA

27th November – 1st December 2017. Mkonge Hotel

Monday, 27th November, 2017 SESSION 1

08.00-09. 00 Registration and Coffee

09.00-09.10 Honourable Engineer Zena Said, the Tanga Regional Administrative Secretary: Welcome speech

09.10-09.20 Adly Abd-Alla and Imna Malele: Introduction and administrative details

09.20-09.50 A. Abd-Alla: Update on research activity on tsetse fly in Seibersdorf: Impact of irradiation on tsetse symbiont, mtDNA and genotyping.

09.50-10.20 De Vooght L., and Van Den Abbeele J.: Paratransgenesis in the tsetse fly: current status and future challenges.

10.20-10.35 COFFEE

SESSION 2

10.35-11.05 Weber, Judith; Waespy, Mario; Nilima Dinesh; Gbem, Thaddeus T; Shaida, Stephen; Thomas Haselhorst; Joe Tiralongo; Mamman, Muhammad; Nok, Jonathan A. and Sorge Kelm.: Sialic acid, trans-

sialidase and sialidase in the midgut of tsetse flies

11.05-11.35 Njiokou F., Kame Ngasse G., Nana-Djeunga H., Melachio-Tanekou T. & Geiger A: Sodalis glossinidius and Wolbachia sp genetic diversities: relation with tsetse fly trypanosome infection in the Faro-Deo AAT in Cameroon.

11.35-13.00 LUNCH

SESSION 3

13.00-13.30 Otto Koekemoer, Chantel de Beer, Jerome Ntshangase. : Comparison of microbial diversity in field vs laboratory reared G. brevipalpis. Skype presentation

13.30-14.00 George Tsiamis, Evangelos Doudoumis, Antonios A. Augustinos, Eva, Dionyssopoulou, Andrew Parker, Adly M.M. Abd-Alla and Kostas Bourtzis : Different laboratory populations similar bacterial profile? The case of Glossina palpalis gambiensiss

14.00-14.30 Solomon Meknnen, Adly M. M. Abd-Alla and Andrew G. Parker : Tissue distribution, Identification and prevalence of symbionts in tsetse flies, Glossina spp. in Ethiopia

14.30-15.00 Tobias Engl, Veronika Michalkova, Brian L. Weiss, Güler D. Uzel, Peter Takac, Wolfgang J. Miller, Adly M. M. Abd-Alla, Serap Aksoy, Martin Kaltenpoth: Effect of antibiotic treatment and gamma-irradiation on cuticular hydrocarbon profiles and mate choice in tsetse flies (Glossina m. morsitans)

15.00-15.15 COFFEE

SESSION 4 15.15-16.45 Daniela I. Schneider and Wolfgang J. Miller: Titer and tissue dynamics of tsetse symbionts in Glossina interspecies hybrids: biological and

applied aspects

16.45-17.15 Abdelaziz Heddi: The bactriome immunity promotes host homeostasis in insect endosymbiosis

17.15-17.45 Brian Weiss, Aurelien Vigneron, Michelle Maltz and Serap Aksoy Colonization of the tsetse fly midgut with commensal Enterobacter inhibits trypanosome infection establishment

17.45-18.30 General discussion

Tuesday, 28th November, 2017

SESSION 5

08:30- 09:00 Grazia Savini, Francesca Scolari, Lino Ometto, Omar Rota-Stabelli, Peter Takac, Geoffrey M. Attardo, Serap Aksoy,Anna R. Malacrida: Male reproductive physiology in the Glossina genus: impact of endosymbionts and gene evolution

09.00-09.30 Emanuel Procházka, Veronika Michalková, Ivana Daubnerová, Ladislav Roller, Dušan Žitňan, Peter Takáč: Molecular genetic approach to reduce fecundity of tsetse flies.

09.30-10.00 Milan Kozánek, Daniel Valaška, Ján Kodada, Peter Takáč: Application of interactive 3D visualization techniques in taxonomic research of Glossinidae.

10.00-10.30 Njelembo J. Mbewe, Samuel Guya , Florence N. Wamwiri: Frequency of maternally-inherited microbes in field-collected Glossina morsitans centralis and association with con-current trypanosome infection

10.30-10.45 COFFEE

SESSION 6

10.45-11.15 Lawrence G Wamiti, Fathiya M Khamis, Adly MM Abd-alla, Fidelis LO Ombura, Komivi S Akutse, Sevgan Subramanian, Sunday Ekesi and Nguya K Maniania: Metarhizium anisopliae infection reduces Trypanosoma congolense multiplication in Glossina fuscipes fuscipes and its ability to acquire or transmit the pathogen.

11.15-11.45 Ouedraogo G.M.S, Demerbass, G, Rayaisse J.B, Traore A., Avgoustinos, A., Parker A., Gimonneau G., Sidibe, I., Ouedraogo, A.G., Traore A., Bayala B.R., Vreysen MJB, Bourtzis K. and Abd-Alla M.M.A.: The prevalence of AAT and HAT causative agents and coinfection dynamics (T. congolense forest type, T. congolense savanna type, T. vivax, T. congolense (Tc), T. evansi, T. avium, T. cruzi, T. simiae, Tsp., T. godefreyi, and T. brucei brucei) in tsetse flies from western Africa.

11.45-12.15 Astan Traore: Dynamic of Glossina after the eradication campaign and tsetse infection rates by trypanosomosis and some symbionts in Mali.

12.15-13.30 LUNCH

SESSION 7

13.30-14.00 Njiokou F, Jacob F2, Melachio TT, Njitchouang GR, Gimonneau G, Abate L, Reveillaud J, Geiger A.: Intestinal Bacterial Communities of Glossina palpalis palpalis from Human African Trypanomiasis Foci in Cameroon.

14.00-14.30 Manun Channumsin, Marc Ciosi*, Dan Masiga, C. Michael R. Turner and Barbara K. Mable: Do Sodalis glossinidius influence the prevalence of trypanosomes in tsetse flies?

14.30-15.00 Malele I, Nyingilili H, Lyaruu E, Tauzin M, Ollivier B, Fardeau M-L, Geiger A: Bacterial diversity obtained by cultural approaches in the gut of G. pallidipes population from the coastal area in Tanzania

15.30-15.45 COFFEE

SESSION 8

15.45-16.15 Meki, I.K., Kariithi, H.M., Parker, A.G., Vreysen M.J.B., Ros, V.I., Vlak, J.M., van Oers, M.M. and Abd-Alla A.M.M. Mechanism of salivary gland hypertrophy virus (SGHV) infections: Prerequisite for trypanosomosis control

16.15-16.45 Meki, I.K., İkbal Agah İnce, Boucias, D. Kariithi, H.M., Parker, A.G., Vreysen M.J.B., Vlak, J.M., van Oers, M.M. and Abd-Alla A.M.M Adly Abd-Alla, Henry M. Kariithi: Involvement of microRNAs in infection of G. pallidipes with Glossina pallidipes salivary gland hypertrophy virus (GpSGHV)"

16.45-17.15 Kariithi,HM, Meki,IK, Vreysen, MJB, Parker,AG, Abd-Alla,AMM: Identification of Cultivable Tsetse Gut Microbiota and Assessment of Their Probiotic Potential to Improve Fly Quality and Performance for SIT

17.15-17.45 Just Vlak: 'Functional genomics of hytrosaviruses: a way forward

17.45-18.30 General discussion

Wednesday 29th November, 2017

SESSION 9

08.30-09.45 General Discussion of the Logical Framework and CRP evaluation

documents and Formation of Two Working Groups (see below)

09.45-10.15 COFFEE

10.15-11.15 General Discussion of the Logical Framework and Formation of Two Working Groups (see below)

11.15-12.00 Working Group Discussions (Group 1 Room xx, Group 2 ROOM xx

12.10-17.00 Excursion to Amboni Caves

17:00- 21:00 Group Dinner- Tanga Beach Resort

Thursday 30th November, 2017

SESSION 10

09.00-09.40 Working Group Discussions

09.45-10.15 COFFEE

10.15-12.30 Working Group Discussions

12.30-13.30 LUNCH

13.30-15.30 Drafting Report

15.30-16.00 COFFEE

16.00-17.00 Drafting Report

Friday 1st December, 2017

SESSION 11

09.00-10.30 Reports of Working Groups and CRP evaluation

10.30-11.00 COFFEE

11.00-12.30 Drafting of CRP evaluation

12.30-13.30 LUNCH

14.00-14.30 General Discussion

Closing Remark by Host Country representative

Working Group 1: Symbionts Weiss, İnce, Heddi, Geiger, Engl, Malacrida, Miller, Njiokou, Tsiamis, Takac, Kozánek Anna

Working Group 2: Pathogens Kariithi, Vlak, Traore, İnce, Kelm, Meki, Malele, Maniania, Ouedraogo, Abbeele, Mekonen, Wamwiri

6. LIST OF PARTICIPANTS

LIST OF PARTICIPANTS TO THE THIRD RCM ON ENHANCING VECTOR REFRACTORINESS TO TRYPANOSOME INFECTION From June 6-10, 2016, Lyon, France

AUSTRALIA UMR INRA/INSA de Lyon BF2I Ms Fleur Ponto* 20, avenue Albert Einstein University of Sydney 69621 Villeurbanne Cedex School of Biologic Sciences Email: [email protected] Sydney, NSW 2006 Email: [email protected] Ms. Anne Geiger UMR 177 IRD-CIRAD AUSTRIA Laboratoire de Recherches et de Coordination Mr Wolfgang Miller sur les Trypanosomoses Medizinische Universität Wien TA A-17 / G Schwarzspanier Str. 17 Campus international de Baillarguet 1090 Vienna Email:[email protected] 34 398 Montpellier cedex 5 (France) Tél: 33.(0)4 67 59 39 25 BELGIUM Fax: 33.(0)4 67 59 38 94 Mr jan van den Abbeele Email: [email protected] Prince Leopold Institute of Tropical Medicine Nationalestraat 155 GERMANY 2000 Antwerpern Mr Engl Tobias Email: [email protected] Max-Planck-Institute for Chemical Ecology Beutenberg Campus BURKINA FASO Hans-Knöll Strasse 8 Ms Gisele Ouedraogo* 07745, Jena Ministere des resources animales Email: [email protected] B.P. 172 Ouagadougou 09 Mr Sorge Kelm Email: [email protected] Universität Bremen Postfach 33 04 40 CAMEROON 28334 Bremen Mr Flobert Njiokou Email: [email protected] Faculté des sciences Université of Yaounde GREECE B.P. 812 Yaounde Mr George Tsiamis Email: [email protected] University of Patras 2 Seferi Street ETHIOPIA 30100 Agrino Mr Solomon Meknon Email: [email protected] NICETT (National Institute for Controlling and Eradication of Tsetse and Trypanosomosis) ITALY P.O. Box 19917 Ms Anna Malacrida Addis Ababa Universita degli Studi di Pavia Email: [email protected] Corso Strada Nuova, 65 27100 Pavia Email: [email protected] FRANCE Mr Abdelaziz Heddi KENYA University of Lyon, INSA-Lyon Ms Florence N. Wamwiri

Kenya Agricultural and Livestock Research [email protected] Organisation KALRI P.O. Box 362 UNITED KINGDOM 00920 Kikuyu Ms Barbara Mable Email: [email protected] University of Glasgow University Gardens Ms F. M. Khamis Glasgow, Scotland G128QW International Centre of Insect Physiology and Email: [email protected] Ecology (ICIPE) P.O. Box 30772, 00100 UNITED REPUBLIC OF TANZANIA Nairobi Ms Imna I. Malele Email: [email protected] Vector & Vector Borne Diseases Research Institute MALI (VVBDRI) Astan Traore P.O. Box 1026, Majani Mapana Laboratoire Central Veterinaire de Bamako Off Korogwe Road Tel 223 76 33 30 72 Email: [email protected] Email [email protected] UNITED STATES OF AMERICA Mr Brian Weiss NETHERLANDS Yale School of Public Health Mr Just Vlak Yale University Laboratory of Virology New Haven, CT 06520 Wageningen University Email: [email protected] Droevendaalsesteeg 1 6708 PB Wageningen Mr Drion Boucias* Email: [email protected] University of Florida Gainesville FL 32611 SOUTH AFRICA Email: [email protected] Mr Otto Koekemoer Agricultural Research Council (ARC) CONSULTANTS Onderstepport Veterinary Institute, Private Bag X05 KENYA 0110 Onderstepoort Mr Henry Kariithi Email: [email protected] Biotechnology Research Institute Kenya Agricultural and Livestock Research Organisation (BRI-KALRO), SLOVAKIA PO Box 57811-00200 Mr Peter Takac Kaptagat Road, Loresho, Nairobi Institute of Zoology Email : [email protected] Slovak Academy of Sciences (SAS) Dubravska cesta 9 842 06 Bratislava Email: [email protected]

Milan Kozánek Email: [email protected]

TURKEY Mr Ikbal Agah Ince ACIBADEM UNIVERSITY School of Medicine Department of Medical Microbiology Atasehir-Istanbul 34752 Email: [email protected]

D-28359 Bremen OBSERVERS Germany Telefone: +49 421 218 63224 GERMANY Mobile: +49 16081110133

Ms Berger Petra. University of Bremen Centre for Bimolecular Interactions Bremen (CBIB) *Participant did not attend the 3rd RCM in AG Prof. Dr. Sørge Kelm Lyon, France Leobener Straße, NW2, Room B2180

7. ANNEX I: WORKING PAPERS

Anna Zaidman-Rémy, Abdelaziz Heddi: Endosymbiont control and load adjustment 53 to insect physiological needs. Francesca Scolari1, Grazia Savini1, Serap Aksoy2, Geoffrey M. Attardo2, Anna R. 57 Malacrida: Exploring the role of endosymbionts in tsetse male reproductive physiology. Njiokou F, Jacob F, Melachio TT, Njitchouang GR, Gimonneau G, Abate L, 61 Reveillaud J, Anne Geiger: Intestinal Bacterial Communities of Glossina palpalis palpalis from Human African Trypanomiasis Foci in Cameroon. Bridget C. Griffith, Brian L. Weiss, Emre Aksoy, Paul O. Mireji, Joana E. Auma, 65 Florence N. Wamwiri, Richard Echodu, Grace Murilla, Serap Aksoy: Analysis of the gut-specific microbiome from field-captured tsetse flies, and its potential relevance to host trypanosome vector competence. Flobert Njiokou, Kame Ngasse G., Farikou O, Melachio Tanekou T.T, Simo, G. 85 Geiger A: Improving knowledge of the interactions between Glossina tachinoides, Glossina morsitans submorsitans, its symbionts Sodalis glosinidus, Wolbachia, and its parasites Trypanosoma sp in the African Animal Trypanosomiasis focus of Adamaoua, northern Cameroon Njelembo J. Mbewe, Cornelius Mweempwa, Samuel Guya and Florence N. 91 Wamwiri: Frequency of Sodalis and Wolbachia in Glossina morsitans centralis and association with trypanosome infection in Western Zambia George Tsiamis, Antonios A. Augustinos, Vangelis Doudoumis, Eva 97 Dionyssopoulou, Peter Takac, Adly M.M. Abd-Alla, Kostas Bourtzis: Detection and Characterization of Bacterial Communities in tsetse flies Imna Malele, Hamisi Nyingilili, Eugene Lyaruu, Ollivier B, Cayol J-L, Fardeau M- 135 L, Anne Geiger: Bacterial diversity obtained by culturable approaches in the gut of Glossina pallidipes population from a non-sleeping sickness focus in Tanzania: preliminary results Linda De Vooght,Guy Caljon & Jan Van Den Abbeele: Update on the Sodalis 157 expression of functional anti-trypanosome nanobodies in different tsetse fly tissues Just M. Vlak, Henry M. Kariithi, Max Bergoin, Irene K. Meki, İkbal A. Ìnce, Adly 165 M.M. Abd-Alla: Glossina species (G. pallipides and G. m. morsitans) with differential hytrosavirus pathologies: A comparison of the salivary gland proteomes. Tobias Engl, Veronika Michalkova, Brian L. Weiss, Güler D. Uzel, Peter Takac, 195 Wolfgang J. Miller, Adly M. M. Abd-Alla, Serap Aksoy, Martin Kaltenpoth: Antibiotic treatment affects cuticular hydrocarbon profiles and mate choice in tsetse flies (Glossina m. morsitans). Wamiti L.G., Fathiya K.H., Ekesi S., Ombura L. and Nguya K. Maniania: Effect of 215 fungal infection by Metarhizium anisopliae on vector competence to transmit Trypanosoma parasite and the effect of fungal infection on fly hemocytes Otto Koekemoer: Microbial diversity in field and colonised Glossina brevipalpis in 217

South Africa Veronika Michalkova, Joshua B. Benoit, Brian L. Weiss, Geoffrey M. Attardo, Serap 225 Aksoy and Peter Takáč: Tsetse fly fertility dependence on nutrient homeostasis and symbiosis Daniela I. Schneider, Andrew G. Parker, Florent Masson, Balmand Séverine, Adly 233 M.M. Abd-Alla, Abdelaziz Heddi, Wolfgang J. Miller: Symbiont-dynamics and transmission modes in tsetse fly hybrids