Tolerance to a Combination of Low Temperature and Sterilizing Irradiation in Male Glossina palpalis gambiensis (Diptera: Glossinidae): Simulated Transport and Release Conditions.

Recently, aerial delivery of sterilized adult tsetse flies has been developed based on the release of chilled adult sterile males. The long-distance transport of irradiated male tsetse pupae for chilled adult release systems requires exposure of the mature pupae to irradiation and to low temperatures for both the pupae and adults. The effect of these treatments on mating of adult Glossina palpalis gambiensis (Vanderplank, Diptera: Glossinidae) males was investigated. Male G. p. gambiensis pupae were stored at 10°C for 5 d and irradiated with 110 Gy within the first 24 h of cold storage. In addition, to simulate a chilled adult release environment, 6-d-old adult males were stored at 5.1 ± 0.4°C for 6 or 30 h. Mating performance was compared to untreated controls in walk-in field cages. A significantly lower proportion of males that had been irradiated and stored at low temperature succeeded in securing a mating compared to untreated males. Premating time, copulation duration and spermathecal fill were similar. Insemination levels were slightly lower for adult males stored at low temperature for 30 h compared to 6 h or control. Although the mating behavior of the males was affected by the treatments given, the data presented confirm the suitability of using long-distance transport of chilled and irradiated male G. p. gambiensis pupae followed by releasing the emerged adult male flies using a chilled adult release system. However, the data indicate that the chilling duration of the adults should be minimized.

Impact of Glossina pallidipes salivary gland hypertrophy virus (GpSGHV) on a heterologous tsetse fly host, Glossina fuscipes fuscipes.

BACKGROUND: Tsetse flies (Diptera: Glossinidae) are the vectors of African trypanosomosis, the causal agent of sleeping sickness in humans and nagana in animals. Glossina fuscipes fuscipes is one of the most important tsetse vectors of sleeping sickness, particularly in Central Africa. Due to the development of resistance of the trypanosomes to the commonly used trypanocidal drugs and the lack of effective vaccines, vector control approaches remain the most effective strategies for sustainable management of those diseases. The Sterile Insect Technique (SIT) is an effective, environment-friendly method for the management of tsetse flies in the context of area-wide integrated pest management programs (AW-IPM). This technique relies on the mass-production of the target insect, its sterilization with ionizing radiation and the release of sterile males in the target area where they will mate with wild females and induce sterility in the native population. It has been shown that Glossina pallidipes salivary gland hypertrophy virus (GpSGHV) infection causes a decrease in fecundity and fertility hampering the maintenance of colonies of the tsetse fly G. pallidipes. This virus has also been detected in different species of tsetse files. In this study, we evaluated the impact of GpSGHV on the performance of a colony of the heterologous host G. f. fuscipes, including the flies' productivity, mortality, survival, flight propensity and mating ability and insemination rates. RESULTS: Even though GpSGHV infection did not induce SGH symptoms, it significantly reduced all examined parameters, except adult flight propensity and insemination rate. CONCLUSION: These results emphasize the important role of GpSGHV management strategy in the maintenance of G. f. fuscipes colonies and the urgent need to implement measures to avoid virus infection, to ensure the optimal mass production of this tsetse species for use in AW-IPM programs with an SIT component.

Combining paratransgenesis with SIT: impact of ionizing radiation on the DNA copy number of Sodalis glossinidius in tsetse flies.

BACKGROUND: Tsetse flies (Diptera: Glossinidae) are the cyclical vectors of the causative agents of African Trypanosomosis, which has been identified as a neglected tropical disease in both humans and animals in many regions of sub-Saharan Africa. The sterile insect technique (SIT) has shown to be a powerful method to manage tsetse fly populations when used in the frame of an area-wide integrated pest management (AW-IPM) program. To date, the release of sterile males to manage tsetse fly populations has only been implemented in areas to reduce transmission of animal African Trypanosomosis (AAT). The implementation of the SIT in areas with Human African Trypanosomosis (HAT) would require additional measures to eliminate the potential risk associated with the release of sterile males that require blood meals to survive and hence, might contribute to disease transmission. Paratransgenesis offers the potential to develop tsetse flies that are refractory to trypanosome infection by modifying their associated bacteria (Sodalis glossinidius) here after referred to as Sodalis. Here we assessed the feasibility of combining the paratransgenesis approach with SIT by analyzing the impact of ionizing radiation on the copy number of Sodalis and the vectorial capacity of sterilized tsetse males. RESULTS: Adult Glossina morsitans morsitans that emerged from puparia irradiated on day 22 post larviposition did not show a significant decline in Sodalis copy number as compared with non-irradiated flies. Conversely, the Sodalis copy number was significantly reduced in adults that emerged from puparia irradiated on day 29 post larviposition and in adults irradiated on day 7 post emergence. Moreover, irradiating 22-day old puparia reduced the copy number of Wolbachia and Wigglesworthia in emerged adults as compared with non-irradiated controls, but the radiation treatment had no significant impact on the vectorial competence of the flies. CONCLUSION: Although the radiation treatment significantly reduced the copy number of some tsetse fly symbionts, the copy number of Sodalis recovered with time in flies irradiated as 22-day old puparia. This recovery offers the opportunity to combine a paratransgenesis approach - using modified Sodalis to produce males refractory to trypanosome infection - with the release of sterile males to minimize the risk of disease transmission, especially in HAT endemic areas. Moreover, irradiation did not increase the vector competence of the flies for trypanosomes.

Hytrosavirus genetic diversity and eco-regional spread in Glossina species.

BACKGROUND: The management of the tsetse species Glossina pallidipes (Diptera; Glossinidae) in Africa by the sterile insect technique (SIT) has been hindered by infections of G. pallidipes production colonies with Glossina pallidipes salivary gland hypertrophy virus (GpSGHV; Hytrosaviridae family). This virus can significantly decrease productivity of the G. pallidipes colonies. Here, we used three highly diverged genes and two variable number tandem repeat regions (VNTRs) of the GpSGHV genome to identify the viral haplotypes in seven Glossina species obtained from 29 African locations and determine their phylogenetic relatedness. RESULTS: GpSGHV was detected in all analysed Glossina species using PCR. The highest GpSGHV prevalence was found in G. pallidipes colonized at FAO/IAEA Insect Pest Control Laboratory (IPCL) that originated from Uganda (100%) and Tanzania (88%), and a lower prevalence in G. morsitans morsitans from Tanzania (58%) and Zimbabwe (20%). Whereas GpSGHV was detected in 25-40% of G. fuscipes fuscipes in eastern Uganda, the virus was not detected in specimens of neighboring western Kenya. Most of the identified 15 haplotypes were restricted to specific Glossina species in distinct locations. Seven haplotypes were found exclusively in G. pallidipes. The reference haplotype H1 (GpSGHV-Uga; Ugandan strain) was the most widely distributed, but was not found in G. swynnertoni GpSGHV. The 15 haplotypes clustered into three distinct phylogenetic clades, the largest contained seven haplotypes, which were detected in six Glossina species. The G. pallidipes-infecting haplotypes H10, H11 and H12 (from Kenya) clustered with H7 (from Ethiopia), which presumably corresponds to the recently sequenced GpSGHV-Eth (Ethiopian) strain. These four haplotypes diverged the most from the reference H1 (GpSGHV-Uga). Haplotypes H1, H5 and H14 formed three main genealogy hubs, potentially representing the ancestors of the 15 haplotypes. CONCLUSION: These data identify G. pallidipes as a significant driver for the generation and diversity of GpSGHV variants. This information may provide control guidance when new tsetse colonies are established and hence, for improved management of the virus in tsetse rearing facilities that maintain multiple Glossina species.

RNA interference-based antiviral immune response against the salivary gland hypertrophy virus in Glossina pallidipes.

BACKGROUND: Glossina pallidipes salivary gland hypertrophy virus (GpSGHV; Hytrosaviridae) is a non-occluded dsDNA virus that specifically infects the adult stages of the hematophagous tsetse flies (Glossina species, Diptera: Glossinidae). GpSGHV infections are usually asymptomatic, but unknown factors can result to a switch to acute symptomatic infection, which is characterized by the salivary gland hypertrophy (SGH) syndrome associated with decreased fecundity that can ultimately lead to a colony collapse. It is uncertain how GpSGHV is maintained amongst Glossina spp. populations but RNA interference (RNAi) machinery, a conserved antiviral defense in insects, is hypothesized to be amongst the host's mechanisms to maintain the GpSGHV in asymptomatic (persistent or latent) infection state. Here, we investigated the involvement of RNAi during GpSGHV infections by comparing the expression of three key RNAi machinery genes, Dicer (DCR), Argonaute (AGO) and Drosha, in artificially virus injected, asymptomatic and symptomatic infected G. pallidipes flies compared to PBS injected (controls) individuals. We further assessed the impact of AGO2 knockdown on virus infection by RT-qPCR quantification of four selected GpSGHV genes, i.e. odv-e66, dnapol, maltodextrin glycosyltransferase (a tegument gene) and SGHV091 (a capsid gene). RESULTS: We show that in response to hemocoelic injections of GpSGHV into G. pallidipes flies, increased virus replication was accompanied by significant upregulation of the expression of three RNAi key genes; AGO1, AGO2 and DCR2, and a moderate increase in the expression of Drosha post injection compared to the PBS-injected controls. Furthermore, compared to asymptomatically infected individuals, symptomatic flies showed significant downregulation of AGO1, AGO2 and Drosha, but a moderate increase in the expression of DCR2. Compared to the controls, knockdown of AGO2 did not have a significant impact on virus infection in the flies as evidenced by unaltered transcript levels of the selected GpSGHV genes. CONCLUSION: The upregulation of the expression of the RNAi genes implicate involvement of this machinery in controlling GpSGHV infections and the establishment of symptomatic GpSGHV infections in Glossina. These findings provide a strategic foundation to understand GpSGHV infections and to control latent (asymptomatic) infections in Glossina spp. and thereby control SGHVs in insect production facilities.

Coevolution of hytrosaviruses and host immune responses.

BACKGROUND: Hytrosaviruses (SGHVs; Hytrosaviridae family) are double-stranded DNA (dsDNA) viruses that cause salivary gland hypertrophy (SGH) syndrome in flies. Two structurally and functionally distinct SGHVs are recognized; Glossina pallidipes SGHV (GpSGHV) and Musca domestica SGHV (MdSGHV), that infect the hematophagous tsetse fly and the filth-feeding housefly, respectively. Genome sizes and gene contents of GpSGHV (~ 190 kb; 160-174 genes) and MdSGHV (~ 124 kb; 108 genes) may reflect an evolution with the SGHV-hosts resulting in differences in pathobiology. Whereas GpSGHV can switch from asymptomatic to symptomatic infections in response to certain unknown cues, MdSGHV solely infects symptomatically. Overt SGH characterizes the symptomatic infections of SGHVs, but whereas MdSGHV induces both nuclear and cellular hypertrophy (enlarged non-replicative cells), GpSGHV induces cellular hyperplasia (enlarged replicative cells). Compared to GpSGHV's specificity to Glossina species, MdSGHV infects other sympatric muscids. The MdSGHV-induced total shutdown of oogenesis inhibits its vertical transmission, while the GpSGHV's asymptomatic and symptomatic infections promote vertical and horizontal transmission, respectively. This paper reviews the coevolution of the SGHVs and their hosts (housefly and tsetse fly) based on phylogenetic relatedness of immune gene orthologs/paralogs and compares this with other virus-insect models. RESULTS: Whereas MdSGHV is not vertically transmitted, GpSGHV is both vertically and horizontally transmitted, and the balance between the two transmission modes may significantly influence the pathogenesis of tsetse virus. The presence and absence of bacterial symbionts (Wigglesworthia and Sodalis) in tsetse and Wolbachia in the housefly, respectively, potentially contributes to the development of SGH symptoms. Unlike MdSGHV, GpSGHV contains not only host-derived proteins, but also appears to have evolutionarily recruited cellular genes from ancestral host(s) into its genome, which, although may be nonessential for viral replication, potentially contribute to the evasion of host's immune responses. Whereas MdSGHV has evolved strategies to counteract both the housefly's RNAi and apoptotic responses, the housefly has expanded its repertoire of immune effector, modulator and melanization genes compared to the tsetse fly. CONCLUSIONS: The ecologies and life-histories of the housefly and tsetse fly may significantly influence coevolution of MdSGHV and GpSGHV with their hosts. Although there are still many unanswered questions regarding the pathogenesis of SGHVs, and the extent to which microbiota influence expression of overt SGH symptoms, SGHVs are attractive 'explorers' to elucidate the immune responses of their hosts, and the transmission modes of other large DNA viruses.

Prevalence of trypanosomes, salivary gland hypertrophy virus and Wolbachia in wild populations of tsetse flies from West Africa.

BACKGROUND: Tsetse flies are vectors of African trypanosomes, protozoan parasites that cause sleeping sickness (or human African trypanosomosis) in humans and nagana (or animal African trypanosomosis) in livestock. In addition to trypanosomes, four symbiotic bacteria Wigglesworthia glossinidia, Sodalis glossinidius, Wolbachia, Spiroplasma and one pathogen, the salivary gland hypertrophy virus (SGHV), have been reported in different tsetse species. 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) that were collected between 2008 and 2015 from 46 geographical locations in West Africa, i.e. Burkina Faso, Mali, Ghana, Guinea, and Senegal. RESULTS: The results indicated an overall low prevalence of SGHV and Wolbachia and a high prevalence of trypanosomes in the sampled wild tsetse populations. The prevalence of all three infections varied among tsetse species and sample origin. The highest trypanosome prevalence was found in Glossina tachinoides (61.1%) from Ghana and in Glossina palpalis gambiensis (43.7%) from Senegal. The trypanosome prevalence in the four species from Burkina Faso was lower, i.e. 39.6% in Glossina medicorum, 18.08%; in Glossina morsitans submorsitans, 16.8%; in Glossina tachinoides and 10.5% in Glossina palpalis gambiensis. The trypanosome prevalence in Glossina palpalis gambiensis was lowest in Mali (6.9%) and Guinea (2.2%). The prevalence of SGHV and Wolbachia was very low irrespective of location or tsetse species with an average of 1.7% for SGHV and 1.0% for Wolbachia. In some cases, mixed infections with different trypanosome species were detected. The highest prevalence of coinfection was Trypanosoma vivax and other Trypanosoma species (9.5%) followed by coinfection of T. congolense with other trypanosomes (7.5%). The prevalence of coinfection of T. vivax and T. congolense was (1.0%) and no mixed infection of trypanosomes, SGHV and Wolbachia was detected. CONCLUSION: The results indicated a high rate of trypanosome infection in tsetse wild populations in West African countries but lower infection rate of both Wolbachia and SGHV. Double or triple mixed trypanosome infections were found. In addition, mixed trypanosome and SGHV infections existed however no mixed infections of trypanosome and/or SGHV with Wolbachia were found.

Mapping landscape friction to locate isolated tsetse populations that are candidates for elimination.

Tsetse flies are the cyclical vectors of deadly human and animal trypanosomes in sub-Saharan Africa. Tsetse control is a key component for the integrated management of both plagues, but local eradication successes have been limited to less than 2% of the infested area. This is attributed to either resurgence of residual populations that were omitted from the eradication campaign or reinvasion from neighboring infested areas. Here we focused on Glossina palpalis gambiensis, a riverine tsetse species representing the main vector of trypanosomoses in West Africa. We mapped landscape resistance to tsetse genetic flow, hereafter referred to as friction, to identify natural barriers that isolate tsetse populations. For this purpose, we fitted a statistical model of the genetic distance between 37 tsetse populations sampled in the region, using a set of remotely sensed environmental data as predictors. The least-cost path between these populations was then estimated using the predicted friction map. The method enabled us to avoid the subjectivity inherent in the expert-based weighting of environmental parameters. Finally, we identified potentially isolated clusters of G. p. gambiensis habitat based on a species distribution model and ranked them according to their predicted genetic distance to the main tsetse population. The methodology presented here will inform the choice on the most appropriate intervention strategies to be implemented against tsetse flies in different parts of Africa. It can also be used to control other pests and to support conservation of endangered species.

Using species distribution models to optimize vector control in the framework of the tsetse eradication campaign in Senegal.

Tsetse flies are vectors of human and animal trypanosomoses in sub-Saharan Africa and are the target of the Pan African Tsetse and Trypanosomiasis Eradication Campaign (PATTEC). Glossina palpalis gambiensis (Diptera: Glossinidae) is a riverine species that is still present as an isolated metapopulation in the Niayes area of Senegal. It is targeted by a national eradication campaign combining a population reduction phase based on insecticide-treated targets (ITTs) and cattle and an eradication phase based on the sterile insect technique. In this study, we used species distribution models to optimize control operations. We compared the probability of the presence of G. p. gambiensis and habitat suitability using a regularized logistic regression and Maxent, respectively. Both models performed well, with an area under the curve of 0.89 and 0.92, respectively. Only the Maxent model predicted an expert-based classification of landscapes correctly. Maxent predictions were therefore used throughout the eradication campaign in the Niayes to make control operations more efficient in terms of deployment of ITTs, release density of sterile males, and location of monitoring traps used to assess program progress. We discuss how the models' results informed about the particular ecology of tsetse in the target area. Maxent predictions allowed optimizing efficiency and cost within our project, and might be useful for other tsetse control campaigns in the framework of the PATTEC and, more generally, other vector or insect pest control programs.

Comprehensive annotation of Glossina pallidipes salivary gland hypertrophy virus from Ethiopian tsetse flies: a proteogenomics approach.

Glossina pallidipes salivary gland hypertrophy virus (GpSGHV; family Hytrosaviridae) can establish asymptomatic and symptomatic infection in its tsetse fly host. Here, we present a comprehensive annotation of the genome of an Ethiopian GpSGHV isolate (GpSGHV-Eth) compared with the reference Ugandan GpSGHV isolate (GpSGHV-Uga; GenBank accession number EF568108). GpSGHV-Eth has higher salivary gland hypertrophy syndrome prevalence than GpSGHV-Uga. We show that the GpSGHV-Eth genome has 190 291 nt, a low G+C content (27.9 %) and encodes 174 putative ORFs. Using proteogenomic and transcriptome mapping, 141 and 86 ORFs were mapped by transcripts and peptides, respectively. Furthermore, of the 174 ORFs, 132 had putative transcriptional signals [TATA-like box and poly(A) signals]. Sixty ORFs had both TATA-like box promoter and poly(A) signals, and mapped by both transcripts and peptides, implying that these ORFs encode functional proteins. Of the 60 ORFs, 10 ORFs are homologues to baculovirus and nudivirus core genes, including three per os infectivity factors and four RNA polymerase subunits (LEF4, 5, 8 and 9). Whereas GpSGHV-Eth and GpSGHV-Uga are 98.1 % similar at the nucleotide level, 37 ORFs in the GpSGHV-Eth genome had nucleotide insertions (n = 17) and deletions (n = 20) compared with their homologues in GpSGHV-Uga. Furthermore, compared with the GpSGHV-Uga genome, 11 and 24 GpSGHV ORFs were deleted and novel, respectively. Further, 13 GpSGHV-Eth ORFs were non-canonical; they had either CTG or TTG start codons instead of ATG. Taken together, these data suggest that GpSGHV-Eth and GpSGHV-Uga represent two different lineages of the same virus. Genetic differences combined with host and environmental factors possibly explain the differential GpSGHV pathogenesis observed in different G. pallidipes colonies.

Effects of Methyl Eugenol Feeding on Mating Compatibility of Asian Population of Bactrocera dorsalis (Diptera: Tephritidae) with African Population and with B. carambolae.

Males of some species included in the Bactrocera dorsalis complex are strongly attracted to methyl eugenol (ME) (1,2-dimethoxy-4-(2-propenyl) benzene), a natural compound occurring in a variety of plant species. ME feeding of males of the B. dorsalis complex is known to enhance their mating competitiveness. Within B. dorsalis, recent studies show that Asian and African populations of B. dorsalis are sexually compatible, while populations of B. dorsalis and Bactrocera carambolae are relatively incompatible. The objectives of this study were to examine whether ME feeding by males affects mating compatibility between Asian and African populations of B. dorsalis and ME feeding reduces male mating incompatibility between B. dorsalis (Asian population) and B. carambolae. The data confirmed that Asian and African populations of B. dorsalis are sexually compatible for mating and showed that ME feeding only increased the number of matings. Though ME feeding also increased the number of matings of B. dorsalis (Asian population) and B. carambolae males but the sexual incompatibility between both species was not reduced by treatment with ME. These results conform to the efforts resolving the biological species limits among B. dorsalis complex and have implications for fruit fly control programs in fields and horticultural trade.

The effects of genetic manipulation, dieldrin treatment and irradiation on the mating competitiveness of male Anopheles arabiensis in field cages.

BACKGROUND: To enable the release of only sterile male Anopheles arabiensis mosquitoes for the sterile insect technique, the genetic background of a wild-type strain was modified to create a genetic sexing strain ANO IPCL1 that was based on a dieldrin resistance mutation. Secondly, the eggs of ANO IPCL1 require treatment with dieldrin to allow complete elimination of female L1 larvae from the production line. Finally, male mosquito pupae need to be treated with an irradiation dose of 75 Gy for sterilization. The effects of these treatments on the competitiveness of male An. arabiensis were studied. METHODS: The competitiveness of ANO IPCL1 males that were treated either with irradiation or both dieldrin and irradiation, was compared with that of the wild-type strain (An. arabiensis Dongola) at a 1:1 ratio in 5.36 m3 semi-field cages located in a climate-controlled greenhouse. In addition, three irradiated: untreated male ratios were tested in semi-field cages (1:1, 5:1 and 10:1) and their competition for virgin wild-type females was assessed. RESULTS: The ANO IPCL1 males were equally competitive as the wild-type males in this semi-field setting. The ANO IPCL1 males irradiated at 75 Gy were approximately half as competitive as the unirradiated wild-type males. ANO IPCL1 males that had been treated with dieldrin as eggs, and irradiated with 75 Gy as pupae were slightly more competitive than males that were only irradiated. Ratios of 1:1, 5:1 and 10:1 irradiated ANO IPCL1 males: untreated wild-type males resulted in 31, 66 and 81% induced sterility in the female cage population, respectively. CONCLUSIONS: An irradiation dose of 75 Gy reduced the competitiveness of male ANO IPCL1 significantly and will need to be compensated by releasing higher numbers of sterile males in the field. However, the dieldrin treatment used to eliminate females appears to have an unexpected radioprotectant effect, however the mechanism is not understood. A sterile to wild-type ratio of 10:1 effectively reduced the population's fertility under the experimental field cage conditions, but further studies in the field will be needed to confirm the efficiency of sterile ANO IPCL1 males when competing against wild males for wild females.

Conserved metallomics in two insect families evolving separately for a hundred million years.

Μetal cofactors are required for enzymatic catalysis and structural stability of many proteins. Physiological metal requirements underpin the evolution of cellular and systemic regulatory mechanisms for metal uptake, storage and excretion. Considering the role of metal biology in animal evolution, this paper asks whether metal content is conserved between different fruit flies. A similar metal homeostasis was previously observed in Drosophilidae flies cultivated on the same larval medium. Each species accumulated in the order of 200 µg iron and zinc and approximately ten-fold less manganese and copper per gram dry weight of the adult insect. In this paper, data on the metal content in fourteen species of Tephritidae, which are major agricultural pests worldwide, are presented. These fruit flies can be polyphagous (e.g., Ceratitis capitata) or strictly monophagous (e.g., Bactrocera oleae) or oligophagous (e.g., Anastrepha grandis) and were maintained in the laboratory on five distinct diets based on olive oil, carrot, wheat bran, zucchini and molasses, respectively. The data indicate that overall metal content and distribution between the Tephritidae and Drosophilidae species was similar. Reduced metal concentration was observed in B. oleae. Feeding the polyphagous C. capitata with the diet of B. oleae resulted in a significant quantitative reduction of all metals. Thus, dietary components affect metal content in some Tephritidae. Nevertheless, although the evidence suggests some fruit fly species evolved preferences in the use or storage of particular metals, no metal concentration varied in order of magnitude between these two families of Diptera that evolved independently for over 100 million years.

Animal trypanosomosis eliminated in a major livestock production region in Senegal following the eradication of a tsetse population.

African animal trypanosomosis (AAT) was one of the main disease-related constraints to the development of intensive livestock production systems in the Niayes region of Senegal, a 30 km wide strip of land along the coast between Dakar and Saint-Louis. To overcome this constraint, the Government of Senegal initiated an area-wide integrated pest management programme combining chemical control tactics with the sterile insect technique to eradicate a population of the tsetse fly Glossina palpalis gambiensis Vanderplank, 1949 (Diptera, Glossinidae) in this area. The project was implemented following a phased conditional approach, and the target area was divided into three blocks treated sequentially. This study aims to assess the temporal dynamics of the prevalence of Trypanosoma spp. during the implementation of this programme. Between 2009 and 2022, 4,359 blood samples were collected from cattle and screened for trypanosomes using both the buffy coat and ELISA techniques, and PCR tests since 2020. The seroprevalence decreased from 18.9% (95%CI: 11.2-26.5) in 2009 to 0% in 2017-2022 in block 1, and from 92.9% (95%CI: 88.2-97) in 2010 to 0% in 2021 in block 2. The parasitological and serological data confirm the entomological monitoring results, i.e., that there is a high probability that the population of G. p. gambiensis has been eradicated from the Niayes and that the transmission of AAT has been interrupted in the treated area. These results indicate the effectiveness of the adopted approach and show that AAT can be sustainably removed through the creation of a zone free of G. p. gambiensis.

Title: Trypanosomose animale éliminée dans une importante région de production d'élevage au Sénégal suite à l'éradication d'une population de glossines. Abstract: La trypanosomose animale africaine (TAA) était l'une des principales contraintes pathologiques au développement de systèmes de production animale intensifs dans les Niayes du Sénégal, une bande de terre large de 30 km longeant la côte entre Dakar et Saint-Louis. Pour surmonter cette contrainte, le Gouvernement du Sénégal a lancé un programme de lutte intégrée à l'échelle de la zone combinant lutte chimique et technique de l'insecte stérile pour éradiquer une population de Glossina palpalis gambiensis Vanderplank, 1949 (Diptera, Glossinidae). Le projet a été mis en œuvre selon une approche conditionnelle progressive, et la zone cible a été divisée en trois blocs, traités de manière séquentielle. L'objectif de cette étude était d'évaluer la dynamique temporelle de la prévalence de Trypanosoma spp. au cours de la mise en œuvre du programme. Entre 2009 et 2022, 4 359 échantillons de sang ont été prélevés sur des bovins et ont fait l'objet d'un dépistage des trypanosomes à l'aide des techniques du buffy-coat et ELISA, ainsi que de test PCR depuis 2020. Dans le bloc 1, la séroprévalence est passée de 18,9 % (IC 95 % : 11,2­26,5) en 2009 à 0 % entre 2017­2022 et de 92,9 % (IC 95 % : 88,2-97) en 2010 à 0 % en 2021 pour le block 2. Les données parasitologiques et sérologiques confirment les résultats du suivi entomologique selon lesquels il est très probable que la population de Glossina palpalis gambiensis soit éradiquée des Niayes, et que la transmission de la TAA a été interrompue dans la zone traitée. Elles indiquent l'efficacité de l'approche adoptée, et montrent que la TAA peut être durablement éliminée grâce à la création d'une zone exempte de G. p. gambiensis.

Development and characterization of microsatellite markers for population genetics of the cocoa pod borer Conopomorpha cramerella (Snellen) (Lepidoptera: Gracillaridae).

The cocoa pod borer (CPB) Conopomorpha cramerella (Snellen) (Lepidoptera: Gracillaridae) is one of the major constraints for cocoa production in South East Asia. In addition to cultural and chemical control methods, autocidal control tactics such as the Sterile Insect Technique (SIT) could be an efficient addition to the currently control strategy, however SIT implementation will depend on the population genetics of the targeted pest. The aim of the present work was to search for suitable microsatellite loci in the genome of CPB that is partially sequenced. Twelve microsatellites were initially selected and used to analyze moths collected from Indonesia, Malaysia, and the Philippines. A quality control verification process was carried out and seven microsatellites found to be suitable and efficient to distinguish differences between CPB populations from different locations. The selected microsatellites were also tested against a closely related species, i.e. the lychee fruit borer Conopomorpha sinensis (LFB) from Vietnam and eight loci were found to be suitable. The availability of these novel microsatellite loci will provide useful tools for the analysis of the population genetics and gene flow of these pests, to select suitable CPB strains to implement the SIT.

Mating harassment may boost the effectiveness of the sterile insect technique for Aedes mosquitoes.

The sterile insect technique is based on the overflooding of a target population with released sterile males inducing sterility in the wild female population. It has proven to be effective against several insect pest species of agricultural and veterinary importance and is under development for Aedes mosquitoes. Here, we show that the release of sterile males at high sterile male to wild female ratios may also impact the target female population through mating harassment. Under laboratory conditions, male to female ratios above 50 to 1 reduce the longevity of female Aedes mosquitoes by reducing their feeding success. Under controlled conditions, blood uptake of females from an artificial host or from a mouse and biting rates on humans are also reduced. Finally, in a field trial conducted in a 1.17 ha area in China, the female biting rate is reduced by 80%, concurrent to a reduction of female mosquito density of 40% due to the swarming of males around humans attempting to mate with the female mosquitoes. This suggests that the sterile insect technique does not only suppress mosquito vector populations through the induction of sterility, but may also reduce disease transmission due to increased female mortality and lower host contact.

Tsetse flies: their biology and control using area-wide integrated pest management approaches.

Tsetse flies are the cyclical vectors of trypanosomes, the causative agents of 'sleeping sickness' or human African trypanosomosis (HAT) in humans and 'nagana' or African animal trypanosomosis (AAT) in livestock in Sub-saharan Africa. Many consider HAT as one of the major neglected tropical diseases and AAT as the single greatest health constraint to increased livestock production. This review provides some background information on the taxonomy of tsetse flies, their unique way of reproduction (adenotrophic viviparity) making the adult stage the only one easily accessible for control, and how their ecological affinities, their distribution and population dynamics influence and dictate control efforts. The paper likewise reviews four control tactics (sequential aerosol technique, stationary attractive devices, live bait technique and the sterile insect technique) that are currently accepted as friendly to the environment, and describes their limitations and advantages and how they can best be put to practise in an IPM context. The paper discusses the different strategies for tsetse control i.e. localised versus area-wide and focusses thereafter on the principles of area-wide integrated pest management (AW-IPM) and the phased-conditional approach with the tsetse project in Senegal as a recent example. We argue that sustainable tsetse-free zones can be created on Africa mainland provided certain managerial and technical prerequisites are in place.

Phytosanitary cold treatment for oranges infested with Bactrocera zonata (Diptera: Tephritidae).

The peach fruit fly, Bactrocera zonata (Saunders), attacks a wide range of tree fruits in countries from Egypt to Vietnam and is occasionally trapped in the United States. Phytosanitary treatments may be required to export fruit hosts of this insect from countries where it is endemic to countries where it is absent but could become established. This research describes comparative studies to determine if B. zonata could be phytosanitarily controlled by cold treatment schedules existing for Ceratitis capitata (Wiedemann) and Anastrepha ludens (Loew), and the development of a cold treatment of 18 d at 1.7 degrees C for B. zonata infesting oranges. Fruit were infested by puncturing holes in oranges and allowing tephritids to oviposit in the holes. The treatments were initiated when the larvae reached late third instar because previous research had shown that stage to be the most cold-tolerant. B. zonata was not found to be confidently as or less cold tolerant than C. capitata; therefore, treatment schedules for the latter are not supported by this research for the former. B. zonata was found to be more susceptible to 1.7 degrees C than A. ludens; therefore, the use of treatment schedules for A. ludens is supported by this research for B. zonata. However, the treatment for A. ludens requires 22 d. A shorter treatment was verified for B. zonata when 36,820 third instars reared from the eggs in oranges were stored at 1.7 degrees C for 18 d with no larvae moving on examination 24 h after removal from the cold treatment chamber.

Development of a novel genetic sexing strain of Ceratitis capitata based on an X-autosome translocation.

Genetic sexing strains (GSS), such as the Ceratitis capitata (medfly) VIENNA 8 strain, facilitate male-only releases and improve the efficiency and cost-effectiveness of sterile insect technique (SIT) applications. Laboratory domestication may reduce their genetic diversity and mating behaviour and hence, refreshment with wild genetic material is frequently needed. As wild males do not carry the T(Y;A) translocation, and wild females do not easily conform to artificial oviposition, the genetic refreshment of this GSS is a challenging and time-consuming process. In the present study, we report the development of a novel medfly GSS, which is based on a viable homozygous T(XX;AA) translocation using the same selectable markers, the white pupae and temperature-sensitive lethal genes. This allows the en masse cross of T(XX;AA) females with wild males, and the backcrossing of F1 males with the T(XX;AA) females thus facilitating the re-establishment of the GSS as well as its genetic refreshment. The rearing efficiency and mating competitiveness of the novel GSS are similar to those of the T(Y;A)-based VIENNA 8 GSS. However, its advantage to easily allow the genetic refreshment is of great importance as it can ensure the mass production of high-quality males and enhanced efficacy of operational SIT programs.

Near-infrared imaging for automated tsetse pupae sex sorting in support of the sterile insect technique.

Tsetse flies are the cyclical vectors of African trypanosomes and one of several methods to manage this vector is the sterile insect technique (SIT). The ability to determine the sex of tsetse pupae with the objective to separate the sexes before adult emergence has been a major goal for decades for tsetse management programmes with an SIT component. Tsetse females develop faster and pharate females inside the pupae melanise 1-2 days before males. This earlier melanisation can be detected by infrared cameras through the pupal shell, and the newly developed Near InfraRed Pupae Sex Sorter (NIRPSS) takes advantage of this. The melanisation process is not homogeneous for all fly organs and the pupa needs to be examined ventrally, dorsally and laterally to ensure accurate classification by an image analysis algorithm. When the pupae are maturing at a constant temperature of 24 °C and sorted at the appropriate age, 24 days post-larviposition for Glossina palpalis gambiensis, the sorting machine can efficiently separate the sexes. The recovered male pupae can then be sterilised for field releases of males, while the rest of the pupae can be used to maintain the laboratory colony. The sorting process with the new NIRPSS had no negative impact on adult emergence and flight ability. A mean male recovery of 62.82 ± 3.61% was enough to provide sterile males to an operational SIT programme, while mean contamination with females (4.69 ± 3.02%) was low enough to have no impact on the maintenance of a laboratory colony.

Title: Imagerie dans l'infrarouge proche pour le tri automatisé du sexe des pupes de glossines comme aide à la technique de l'insecte stérile. Abstract: Les glossines sont les vecteurs cycliques des trypanosomes africains et la technique de l'insecte stérile (TIS) est l'une des méthodes de gestion de ce vecteur. La capacité à déterminer le sexe des pupes de glossines dans le but de séparer les sexes avant l'émergence des adultes a été un objectif majeur, pendant des décennies, pour les programmes de lutte contre les glossines avec une composante TIS. Les femelles tsé-tsé se développent plus rapidement et les pharates femelles à l'intérieur des pupes se mélanisent 1 à 2 jours avant les mâles. Cette mélanisation précoce peut être détectée par des caméras infrarouges à travers la coque de la pupe, ce que le nouveau trieur de sexe des pupes dans le proche infrarouge (TSPPIR) utilise. Le processus de mélanisation n'est pas homogène pour tous les organes de la mouche et la pupe doit être examinée ventralement, dorsalement et latéralement pour assurer une classification précise par un algorithme d'analyse d'image. Lorsque les pupes mûrissent à une température constante de 24 °C et sont triées à l'âge approprié, 24 jours après la larviposition pour Glossina palpalis gambiensis, la machine de tri peut séparer efficacement les sexes. Les pupes mâles récupérées peuvent ensuite être stérilisées pour les lâchers de mâles sur le terrain tandis que le reste des pupes peut être utilisé pour maintenir la colonie de laboratoire. Le processus de tri avec le nouveau TSPPIR n'a eu aucun impact négatif sur l'émergence et la capacité de vol des adultes. Une récupération moyenne des mâles de 62,82 ± 3,61% était suffisante pour fournir des mâles stériles à un programme TIS opérationnel, tandis que la contamination moyenne par les femelles (4,69 ± 3,02%) était suffisamment faible pour n'avoir aucun impact sur le maintien d'une colonie de laboratoire.