The internal transcribed spacer 1 sequence polymorphism brings updates to tsetse species distribution in the northern Cameroon: Importance in planning efficient vector control.

Vector control remains one of the best strategies to prevent the transmission of trypanosome infections in humans and livestock and, thus, a good way to achieve the elimination of human African trypanosomiasis and animal African trypanosomiasis. A key prerequisite for the success of any vector control strategy is the accurate identification and correct mapping of tsetse species. In this work, we updated the tsetse fly species identification and distribution in many geographical areas in Cameroon. Tsetse flies were captured from six localities in Cameroon, and their species were morphologically identified. Thereafter, DNA was extracted from legs of each tsetse fly and the length polymorphism of internal transcribed spacer-1 (ITS1) region of each fly was investigated using PCR. ITS1 DNA fragments of each tsetse species were sequenced. The sequences obtained were analysed and compared to those available in GenBank. This enabled to confirm/infirm results of the morphologic identification and then, to establish the phylogenetic relationships between tsetse species. Morphologic features allowed to clearly distinguish all the tsetse species captured in the South Region of Cameroon, that is, Glossina palpalis palpalis, G. pallicera, G. caliginea and G. nigrofusca. In the northern area, G. morsitans submorsitans could also be distinguished from G. palpalis palpalis, G. tachinoides and G. fuscipes, but these three later could not be distinguished with routine morphological characters. The ITS1 length polymorphism was high among most of the studied species and allowed to identify the following similar species with a single PCR, that is, G. palpalis palpalis with 241 or 242 bp and G. tachinoides with 221 or 222 bp, G. fuscipes with 236 or 237 bp. We also updated the old distribution of tsetse species in the areas assessed, highlighting the presence of G. palpalis palpalis instead of G. fuscipes in Mbakaou, or in sympatry with G. morsitans submorsitans in Dodeo (northern Cameroon). This study confirms the presence of G. palpalis palpalis in the Adamawa Region of Cameroon. It highlights the limits of using morphological criteria to differentiate some tsetse species. Molecular tools based on the polymorphism of ITS1 of tsetse flies can differentiate tsetse species through a simple PCR before downstream analyses or vector control planning.

Infection with endosymbiotic Spiroplasma disrupts tsetse (Glossina fuscipes fuscipes) metabolic and reproductive homeostasis.

Tsetse flies (Glossina spp.) house a population-dependent assortment of microorganisms that can include pathogenic African trypanosomes and maternally transmitted endosymbiotic bacteria, the latter of which mediate numerous aspects of their host's metabolic, reproductive, and immune physiologies. One of these endosymbionts, Spiroplasma, was recently discovered to reside within multiple tissues of field captured and laboratory colonized tsetse flies grouped in the Palpalis subgenera. In various arthropods, Spiroplasma induces reproductive abnormalities and pathogen protective phenotypes. In tsetse, Spiroplasma infections also induce a protective phenotype by enhancing the fly's resistance to infection with trypanosomes. However, the potential impact of Spiroplasma on tsetse's viviparous reproductive physiology remains unknown. Herein we employed high-throughput RNA sequencing and laboratory-based functional assays to better characterize the association between Spiroplasma and the metabolic and reproductive physiologies of G. fuscipes fuscipes (Gff), a prominent vector of human disease. Using field-captured Gff, we discovered that Spiroplasma infection induces changes of sex-biased gene expression in reproductive tissues that may be critical for tsetse's reproductive fitness. Using a Gff lab line composed of individuals heterogeneously infected with Spiroplasma, we observed that the bacterium and tsetse host compete for finite nutrients, which negatively impact female fecundity by increasing the length of intrauterine larval development. Additionally, we found that when males are infected with Spiroplasma, the motility of their sperm is compromised following transfer to the female spermatheca. As such, Spiroplasma infections appear to adversely impact male reproductive fitness by decreasing the competitiveness of their sperm. Finally, we determined that the bacterium is maternally transmitted to intrauterine larva at a high frequency, while paternal transmission was also noted in a small number of matings. Taken together, our findings indicate that Spiroplasma exerts a negative impact on tsetse fecundity, an outcome that could be exploited for reducing tsetse population size and thus disease transmission.

Developments in the classification and nomenclature of arthropod-infecting large DNA viruses that contain pif genes.

Viruses of four families of arthropod-specific, large dsDNA viruses (the nuclear arthropod large DNA viruses, or NALDVs) possess homologs of genes encoding conserved components involved in the baculovirus primary infection mechanism. The presence of such homologs encoding per os infectivity factors (pif genes), along with their absence from other viruses and the occurrence of other shared characteristics, suggests a common origin for the viruses of these families. Therefore, the class Naldaviricetes was recently established, accommodating these four families. In addition, within this class, the ICTV approved the creation of the order Lefavirales for three of these families, whose members carry homologs of the baculovirus genes that code for components of the viral RNA polymerase, which is responsible for late gene expression. We further established a system for the binomial naming of all virus species in the order Lefavirales, in accordance with a decision by the ICTV in 2019 to move towards a standardized nomenclature for all virus species. The binomial species names for members of the order Lefavirales consist of the name of the genus to which the species belongs (e.g., Alphabaculovirus), followed by a single epithet that refers to the host species from which the virus was originally isolated. The common names of viruses and the abbreviations thereof will not change, as the format of virus names lies outside the remit of the ICTV.

Viviparity and habitat restrictions may influence the evolution of male reproductive genes in tsetse fly (Glossina) species.

BACKGROUND: Glossina species (tsetse flies), the sole vectors of African trypanosomes, maintained along their long evolutionary history a unique reproductive strategy, adenotrophic viviparity. Viviparity reduces their reproductive rate and, as such, imposes strong selective pressures on males for reproductive success. These species live in sub-Saharan Africa, where the distributions of the main sub-genera Fusca, Morsitans, and Palpalis are restricted to forest, savannah, and riverine habitats, respectively. Here we aim at identifying the evolutionary patterns of the male reproductive genes of six species belonging to these three main sub-genera. We then interpreted the different patterns we found across the species in the light of viviparity and the specific habitat restrictions, which are known to shape reproductive behavior. RESULTS: We used a comparative genomic approach to build consensus evolutionary trees that portray the selective pressure acting on the male reproductive genes in these lineages. Such trees reflect the long and divergent demographic history that led to an allopatric distribution of the Fusca, Morsitans, and Palpalis species groups. A dataset of over 1700 male reproductive genes remained conserved over the long evolutionary time scale (estimated at 26.7 million years) across the genomes of the six species. We suggest that this conservation may result from strong functional selective pressure on the male imposed by viviparity. It is noteworthy that more than half of these conserved genes are novel sequences that are unique to the Glossina genus and are candidates for selection in the different lineages. CONCLUSIONS: Tsetse flies represent a model to interpret the evolution and differentiation of male reproductive biology under different, but complementary, perspectives. In the light of viviparity, we must take into account that these genes are constrained by a post-fertilization arena for genomic conflicts created by viviparity and absent in ovipositing species. This constraint implies a continuous antagonistic co-evolution between the parental genomes, thus accelerating inter-population post-zygotic isolation and, ultimately, favoring speciation. Ecological restrictions that affect reproductive behavior may further shape such antagonistic co-evolution.

ICTV Virus Taxonomy Profile: Hytrosaviridae.

Hytrosaviridae is a family of large, rod-shaped, enveloped entomopathogenic viruses with dsDNA genomes of 120-190 kbp. Hytrosaviruses (also known as salivary gland hypertrophy viruses) primarily replicate in the salivary glands of adult dipteran flies. Hytrosaviruses infecting the haematophagous tsetse fly and the filth-feeding housefly are assigned to two genera, Glossinavirus and Muscavirus, respectively. Whereas muscavirus infections are only overt, glossinavirus infections can be either covert or overt. Overt infections are characterized by diagnostic salivary gland hypertrophy and cause either partial or complete infertility. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Hytrosaviridae, which is available at ictv.global/report/hytrosaviridae.

Blood feeding tsetse flies as hosts and vectors of mammals-pre-adapted African Trypanosoma: current and expected research directions.

Research on the zoo-anthropophilic blood feeding tsetse flies' biology conducted, by different teams, in laboratory settings and at the level of the ecosystems- where also co-perpetuate African Trypanosoma- has allowed to unveil and characterize key features of tsetse flies' bacterial symbionts on which rely both (a) the perpetuation of the tsetse fly populations and (b) the completion of the developmental program of the African Trypanosoma. Transcriptomic analyses have already provided much information on tsetse fly genes as well as on genes of the fly symbiotic partners Sodalis glossinidius and Wigglesworthia, which account for the successful onset or not of the African Trypanosoma developmental program. In parallel, identification of the non- symbiotic bacterial communities hosted in the tsetse fly gut has recently been initiated: are briefly introduced those bacteria genera and species common to tsetse flies collected from distinct ecosystems, that could be further studied as potential biologicals preventing the onset of the African Trypanosoma developmental program. Finally, future work will need to concentrate on how to render tsetse flies refractory, and the best means to disseminate them in the field in order to establish an overall refractory fly population.

High-sensitivity detection of cryptic Wolbachia in the African tsetse fly (Glossina spp.).

BACKGROUND: In African tsetse flies Glossina, spp. detection of bacterial symbionts such as Wolbachia is challenging since their prevalence and distribution are patchy, and natural symbiont titers can range at levels far below detection limit of standard molecular techniques. Reliable estimation of symbiont infection frequency, especially with regard to interrelations between symbionts and their potential impact on host biology, is of pivotal interest in the context of future applications for the control and eradication of Glossina-vectored African trypanosomosis. The presence or absence of symbionts is routinely screened with endpoint polymerase chain reaction (PCR), which has numerous advantages, but reaches its limits, when detecting infections at natural low titer. To not only determine presence of native tsetse symbionts but also to localize them to specific host tissues, fluorescence in situ hybridization (FISH) can be applied. However, classic FISH assays may not detect low-titer infections due to limitations in sensitivity. RESULTS: We have compared classic endpoint PCR with high-sensitivity blot-PCR. We demonstrate that the latter technique allows for clear detection of low-titer Wolbachia in the morsitans and palpalis groups while classic endpoint PCR does not. In order to localize Wolbachia in situ in high and low-titer Glossina species, we applied high-end Stellaris® rRNA-FISH. We show that with this high sensitivity method, even low amounts of Wolbachia can be traced in specific tissues. Furthermore, we highlight that more tissues and organs than previously recorded are infested with Wolbachia in subspecies of the morsitans and palpalis groups. CONCLUSIONS: Our results demonstrate that overall symbiont infection frequencies as well as the presence in specific host tissues may be underestimated when using low-sensitivity methods. To better understand the complex interrelation of tsetse flies and their native symbionts plus the pathogenic trypanosomes, it is important to consider application of a broader range of high-sensitivity detection tools.

Different laboratory populations similar bacterial profile? The case of Glossina palpalis gambiensis.

BACKGROUND: Microbiota plays an important role in the biology, ecology and evolution of insects including tsetse flies. The bacterial profile of 3 Glossina palpalis gambiensis laboratory colonies was examined using 16S rRNA gene amplicon sequencing to evaluate the dynamics of the bacterial diversity within and between each G. p. gambiensis colony. RESULTS: 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 statistically significant differences were observed between testes and ovaries. Wigglesworthia and Sodalis were the most dominant taxa. In more detail, the gastrointestinal tract of adults was 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. CONCLUSIONS: Our results indicated that age, gender and the origin of the laboratory colonies did not significantly influence the formation of the bacterial profile, once these populations were kept under the same rearing conditions. Within the laboratory populations a tissue tropism was observed between the gut and gonadal bacterial profile.

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.

Effect of antibiotic treatment and gamma-irradiation on cuticular hydrocarbon profiles and mate choice in tsetse flies (Glossina m. morsitans).

BACKGROUND: Symbiotic microbes represent a driving force of evolutionary innovation by conferring novel ecological traits to their hosts. Many insects are associated with microbial symbionts that contribute to their host's nutrition, digestion, detoxification, reproduction, immune homeostasis, and defense. In addition, recent studies suggest a microbial involvement in chemical communication and mating behavior, which can ultimately impact reproductive isolation and, hence, speciation. Here we investigated whether a disruption of the microbiota through antibiotic treatment or irradiation affects cuticular hydrocarbon profiles, and possibly mate choice behavior in the tsetse fly, Glossina morsitans morsitans. Four independent experiments that differentially knock down the multiple bacterial symbionts of tsetse flies were conducted by subjecting tsetse flies to ampicillin, tetracycline, or gamma-irradiation and analyzing their cuticular hydrocarbon profiles in comparison to untreated controls by gas chromatography - mass spectrometry. In two of the antibiotic experiments, flies were mass-reared, while individual rearing was done for the third experiment to avoid possible chemical cross-contamination between individual flies. RESULTS: All three antibiotic experiments yielded significant effects of antibiotic treatment (particularly tetracycline) on cuticular hydrocarbon profiles in both female and male G. m. morsitans, while irradiation itself had no effect on the CHC profiles. Importantly, tetracycline treatment reduced relative amounts of 15,19,23-trimethyl-heptatriacontane, a known compound of the female contact sex pheromone, in two of the three experiments, suggesting a possible implication of microbiota disturbance on mate choice decisions. Concordantly, both female and male flies preferred non-treated over tetracycline-treated flies in direct choice assays. CONCLUSIONS: While we cannot exclude the possibility that antibiotic treatment had a directly detrimental effect on fly vigor as we are unable to recolonize antibiotic treated flies with individual symbiont taxa, our results are consistent with an effect of the microbiota, particularly the obligate nutritional endosymbiont Wigglesworthia, on CHC profiles and mate choice behavior. These findings highlight the importance of considering host-microbiota interactions when studying chemical communication and mate choice in insects.

Nuclear and Wolbachia-based multimarker approach for the rapid and accurate identification of tsetse species.

BACKGROUND: Tsetse flies (Diptera: Glossinidae) are solely responsible for the transmission of African trypanosomes, causative agents of sleeping sickness in humans and nagana in livestock. Due to the lack of efficient vaccines and the emergence of drug resistance, vector control approaches such as the sterile insect technique (SIT), remain the most effective way to control disease. SIT is a species-specific approach and therefore requires accurate identification of natural pest populations at the species level. However, the presence of morphologically similar species (species complexes and sub-species) in tsetse flies challenges the successful implementation of SIT-based population control. RESULTS: In this study, we evaluate different molecular tools that can be applied for the delimitation of different Glossina species using tsetse samples derived from laboratory colonies, natural populations and museum specimens. The use of mitochondrial markers, nuclear markers (including internal transcribed spacer 1 (ITS1) and different microsatellites), and bacterial symbiotic markers (Wolbachia infection status) in combination with relatively inexpensive techniques such as PCR, agarose gel electrophoresis, and to some extent sequencing provided a rapid, cost effective, and accurate identification of several tsetse species. CONCLUSIONS: The effectiveness of SIT benefits from the fine resolution of species limits in nature. The present study supports the quick identification of large samples using simple and cost effective universalized protocols, which can be easily applied by countries/laboratories with limited resources and expertise.

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.

Metarhizium anisopliae infection reduces Trypanosoma congolense reproduction in Glossina fuscipes fuscipes and its ability to acquire or transmit the parasite.

BACKGROUND: Tsetse fly-borne trypanosomiasis remains a significant problem in Africa despite years of interventions and research. The need for new strategies to control and possibly eliminate trypanosomiasis 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 flies and ticks, which may subsequently impact the development and transmission of parasites. Here, we report on the effects of infection of tsetse fly (Glossina fuscipes fuscipes) by the EPF, Metarhizium anisopliae ICIPE 30 wild-type strain (WT) and green fluorescent protein-transformed strain (GZP-1) on the ability of the flies to harbor and transmit the parasite, Trypanosoma congolense. RESULTS: Teneral flies were fed T. congolense-infected blood for 2 h and then infected using velvet carpet fabric impregnated with conidia covered inside a cylindrical plastic tube for 12 h. Control flies were fed with T. congolense-infected blood but not exposed to the fungal treatment via the carpet fabric inside a cylindrical plastic tube. Insects were dissected at 2, 3, 5 and 7 days post-fungal exposure and the density of parasites quantified. Parasite load decreased from 8.7 × 107 at day 2 to between 8.3 × 104 and 1.3 × 105 T. congolense ml- 1 at day 3 post-fungal exposure in fungus-treated (WT and GZP-1) fly groups. When T. congolense-infected flies were exposed to either fungal strain, they did not transmit the parasite to mice whereas control treatment flies remained capable of parasite transmission. Furthermore, M. anisopliae-inoculated flies which fed on T. congolense-infected mice were not able to acquire the parasites at 4 days post-fungal exposure while parasite acquisition was observed in the control treatment during the same period. CONCLUSIONS: Infection of the vector G. f. fuscipes by the entomopathogenic fungus M. anisopliae negatively affected the multiplication of the parasite T. congolense in the fly and reduced the vectorial capacity to acquire or transmit the parasite.

Enhancing vector refractoriness to trypanosome infection: achievements, challenges and perspectives.

With the absence of effective prophylactic vaccines and drugs against African trypanosomosis, control of this group of zoonotic neglected tropical diseases depends the control of the tsetse fly vector. When applied in an area-wide insect pest management approach, the sterile insect technique (SIT) is effective in eliminating single tsetse species from isolated populations. The need to enhance the effectiveness of SIT led to the concept of investigating tsetse-trypanosome interactions by a consortium of researchers in a five-year (2013-2018) Coordinated Research Project (CRP) organized by the Joint Division of FAO/IAEA. The goal of this CRP was to elucidate tsetse-symbiome-pathogen molecular interactions to improve SIT and SIT-compatible interventions for trypanosomoses control by enhancing vector refractoriness. This would allow extension of SIT into areas with potential disease transmission. This paper highlights the CRP's major achievements and discusses the science-based perspectives for successful mitigation or eradication of African trypanosomosis.

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.

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.