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Tsetse flies (Diptera: Glossinidae) are vectors of the tropical neglected diseases sleeping sickness in humans and nagana in animals. The elimination of these diseases is linked to control of the vector. The sterile insect technique (SIT) is an environment-friendly method that has been shown to be effective when applied in an area-wide integrated pest management approach. However, as irradiated males conserve their vectorial competence, there is the potential risk of trypanosome transmission with their release in the field. Analyzing the interaction between the tsetse fly and its microbiota, and between different microbiota and the trypanosome, might provide important information to enhance the fly’s resistance to trypanosome infection. This study on the prevalence of Spiroplasma in wild populations of seven tsetse species from East, West, Central and Southern Africa showed that Spiroplasma is present only in Glossina fuscipes fuscipes and Glossina tachinoides . In G. tachinoides, a significant deviation from independence in co-infection with Spiroplasma and Trypanosoma spp. was observed. Moreover, Spiroplasma infections seem to significantly reduce the density of the trypanosomes, suggesting that Spiroplasma might enhance tsetse fly’s refractoriness to the trypanosome infections. This finding might be useful to reduce risks associated with the release of sterile males during SIT implementation in trypanosome endemic areas. Les mouches tsé-tsé (Diptera : Glossinidae) sont les vecteurs de maladies tropicales négligées, la maladie du sommeil chez l’homme et la nagana chez les animaux. L’élimination de ces maladies est liée à la lutte contre le vecteur. La technique de l’insecte stérile (TIS) est une méthode respectueuse de l’environnement qui s’est révélée efficace lorsqu’elle est appliquée dans le cadre d’une approche de lutte antiparasitaire intégrée à l’échelle d’une zone. Cependant, comme les mâles irradiés conservent leur compétence vectorielle, il existe un risque potentiel de transmission des trypanosomes lors de la libération des mâles sur le terrain. L’analyse de l’interaction entre la mouche tsé-tsé et son microbiote, et entre différents microbiotes et le trypanosome, pourrait fournir des informations importantes pour améliorer la résistance de la mouche à l’infection trypanosomienne. Cette étude sur la prévalence de Spiroplasma dans les populations sauvages de sept espèces de glossines d’Afrique de l’Est, de l’Ouest, centrale et australe a montré que Spiroplasma est présent uniquement chez Glossina fuscipes fuscipes et Glossina tachinoides . Chez G. tachinoides , un écart significatif par rapport à l’indépendance dans la co-infection par Spiroplasma et Trypanosoma spp. a été observé. De plus, les infections à Spiroplasma semblent réduire considérablement la densité des trypanosomes, ce qui suggère que Spiroplasma pourrait renforcer le caractère réfractaire de la mouche tsé-tsé aux infections trypanosomiennes. Cette découverte pourrait être utile pour réduire le risque associé à la libération de mâles stériles lors de la mise en œuvre de la TIS dans les zones d’endémie trypanosomienne.

BACKGROUND: Hytrosaviridae cause salivary gland hypertrophy (SGH) syndrome in some infected tsetse flies (Diptera: Glossinidae). Infected male and female G. pallidipes with SGH have a reduced fecundity and fertility. Due to the deleterious impact of the virus on G. pallidipes colonies, adding the antiviral drug valacyclovir to the blood diet and changing the feeding regime to a clean feeding system (each fly receives for each feeding a fresh clean blood meal) have been investigated to develop virus management strategies. Although both approaches used alone successfully reduced the virus load and the SGH prevalence in small experimental groups, considerable time was needed to obtain the desired SGH reduction and both systems were only demonstrated with colonies that had a low initial virus prevalence (SGH ≤ 10%). As problems with SGH are often only recognized once the incidence is already high, it was necessary to demonstrate that this combination would also work for high prevalence colonies. FINDINGS: Combining both methods at colony level successfully suppressed the SGH in G. pallidipes colonies that had a high initial virus prevalence (average SGH of 24%). Six months after starting the combined treatment SGH symptoms were eliminated from the treated colony, in contrast to 28 months required to obtain the same results using clean feeding alone and 21 months using antiviral drug alone. CONCLUSIONS: Combining valacyclovir treatment with the clean feeding system provides faster control of SGH in tsetse than either method alone and is effective even when the initial SGH prevalence is high.

Tsetse flies are cyclic vectors of Trypanosoma parasites, which cause debilitating diseases in humans and animals. To decrease the disease burden, the number of flies is reduced using the sterile insect technique (SIT), where male flies are sterilized through irradiation and released into the field. This procedure requires the mass rearing of high-quality male flies able to compete with wild male flies for mating with wild females. Recently, two RNA viruses, an iflavirus and a negevirus, were discovered in mass-reared Glossina morsitans morsitans and named GmmIV and GmmNegeV, respectively. The aim of this study was to evaluate whether the densities of these viruses in tsetse flies are affected by the irradiation treatment. Therefore, we exposed tsetse pupae to various doses (0–150 Gy) of ionizing radiation, either in air (normoxia) or without air (hypoxia), for which oxygen was displaced by nitrogen. Pupae and/or emerging flies were collected immediately afterwards, and at three days post irradiation, virus densities were quantified through RT-qPCR. Generally, the results show that irradiation exposure had no significant impact on the densities of GmmIV and GmmNegeV, suggesting that the viruses are relatively radiation-resistant, even at higher doses. However, sampling over a longer period after irradiation would be needed to verify that densities of these insect viruses are not changed by the sterilisation treatment.

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.

The Mediterranean fruit fly, Ceratitis capitata, is one of the most serious pests of fruit crops world-wide. During the last decades, area-wide pest management (AW-IPM) approaches with a sterile insect technique (SIT) component have been used to control populations of this pest in an effective and environment-friendly manner. The development of genetic sexing strains (GSS), such as the Vienna 8 strain, has been played a major role in increasing the efficacy and reducing the cost of SIT programs. However, mass rearing, extensive inbreeding, possible bottleneck phenomena and hitch-hiking effects might pose major risks for deterioration and loss of important genetic characteristics of domesticated insect. In the present study, we present a modified procedure to cryopreserve the embryos of the medfly Vienna 8 GSS based on vitrification and used this strain as insect model to assess the impact of the cryopreservation process on the genetic structure of the cryopreserved insects. Forty-eight hours old embryos, incubated at 24°C, were found to be the most suitable developmental stage for cryopreservation treatment for high production of acceptable hatch rate (38%). Our data suggest the absence of any negative impact of the cryopreservation process on egg hatch rate, pupation rates, adult emergence rates and stability of the temperature sensitive lethal (tsl) character on two established cryopreserved lines (flies emerged from cryopreserved embryos), named V8-118 and V8-228. Taken together, our study provides an optimized procedure to cryopreserve the medfly Vienna 8 GSS and documents the absence of any negative impact on the genetic structure and quality of the strain. Benefits and sceneries for utilization of this technology to support operational SIT projects are discussed in this paper.

BACKGROUND: Dengue viruses (DENVs) are mosquito-borne viruses which can cause disease ranging from mild fever to severe dengue infection. These viruses are endemic in several tropical and subtropical regions. Multiple outbreaks of DENV serotypes 1, 2 and 3 (DENV-1, DENV-2 and DENV-3) have been reported from the western region in Saudi Arabia since 1994. Strains from at least two genotypes of DENV-1 (Asia and America/Africa genotypes) have been circulating in western Saudi Arabia until 2006. However, all previous studies reported from Saudi Arabia were based on partial sequencing data of the envelope (E) gene without any reports of full genome sequences for any DENV serotypes circulating in Saudi Arabia. FINDINGS: Here, we report the isolation and the first complete genome sequence of a DENV-1 strain (DENV-1-Jeddah-1-2011) isolated from a patient from Jeddah, Saudi Arabia in 2011. Whole genome sequence alignment and phylogenetic analysis showed high similarity between DENV-1-Jeddah-1-2011 strain and D1/H/IMTSSA/98/606 isolate (Asian genotype) reported from Djibouti in 1998. Further analysis of the full envelope gene revealed a close relationship between DENV-1-Jeddah-1-2011 strain and isolates reported between 2004–2006 from Jeddah as well as recent isolates from Somalia, suggesting the widespread of the Asian genotype in this region. CONCLUSIONS: These data suggest that strains belonging to the Asian genotype might have been introduced into Saudi Arabia long before 2004 most probably by African pilgrims and continued to circulate in western Saudi Arabia at least until 2011. Most importantly, these results indicate that pilgrims from dengue endemic regions can play an important role in the spread of new DENVs in Saudi Arabia and the rest of the world. Therefore, availability of complete genome sequences would serve as a reference for future epidemiological studies of DENV-1 viruses.

MANY SPECIES OF TSETSE FLIES (DIPTERA: Glossinidae) are infected with a virus that causes salivary gland hypertrophy (SGH), and flies with SGH symptoms have a reduced fecundity and fertility. The prevalence of SGH in wild tsetse populations is usually very low (0.2%-5%), but higher prevalence rates (15.2%) have been observed occasionally. The successful eradication of a Glossina austeni population from Unguja Island (Zanzibar) using an area-wide integrated pest management approach with a sterile insect technique (SIT) component (1994-1997) encouraged several African countries, including Ethiopia, to incorporate the SIT in their national tsetse control programs. A large facility to produce tsetse flies for SIT application in Ethiopia was inaugurated in 2007. To support this project, a Glossina pallidipes colony originating from Ethiopia was successfully established in 1996, but later up to 85% of adult flies displayed symptoms of SGH. As a result, the colony declined and became extinct by 2002. The difficulties experienced with the rearing of G. pallidipes, epitomized by the collapse of the G. pallidipes colony originating from Ethiopia, prompted the urgent need to develop management strategies for the salivary gland hypertrophy virus (SGHV) for this species. As a first step to identify suitable management strategies, the virus isolated from G. pallidipes (GpSGHV) was recently sequenced and research was initiated on virus transmission and pathology. Different approaches to prevent virus replication and its horizontal transmission during blood feeding have been proposed. These include the use of antiviral drugs such as acyclovir and valacyclovir added to the blood for feeding or the use of antibodies against SGHV virion proteins. In addition, preliminary attempts to silence the expression of an essential viral protein using RNA interference will be discussed.

Many species of tsetse flies are infected with a virus that causes salivary gland hypertrophy (SGH) symptoms associated with a reduced fecundity and fertility. A high prevalence of SGH has been correlated with the collapse of two laboratory colonies of Glossina pallidipes and colony maintenance problems in a mass rearing facility in Ethiopia. Mass-production of G. pallidipes is crucial for programs of tsetse control including the sterile insect technique (SIT), and therefore requires a management strategy for this virus. Based on the homology of DNA polymerase between salivary gland hypertrophy virus and herpes viruses at the amino acid level, two antiviral drugs, valacyclovir and acyclovir, classically used against herpes viruses were selected and tested for their toxicity on tsetse flies and their impact on virus replication. While long term per os administration of acyclovir resulted in a significant reduction of productivity of the colonies, no negative effect was observed in colonies fed with valacyclovir-treated blood. Furthermore, treatment of a tsetse colony with valacyclovir for 83 weeks resulted in a significant reduction of viral loads and consequently suppression of SGH symptoms. The combination of initial selection of SGHV-negative flies by non-destructive PCR, a clean feeding system, and valacyclovir treatment resulted in a colony that was free of SGH syndromes in 33 weeks. This is the first report of the use of a drug to control a viral infection in an insect and of the demonstration that valacyclovir can be used to suppress SGH in colonies of G. pallidipes.

Salivary gland hypertrophy viruses (SGHVs) have been identified from different dipteran species, such as the tsetse fly Glossina pallidipes (GpSGHV), the housefly Musca domestica (MdSGHV) and the narcissus bulbfly Merodon equestris (MeSGHV). These viruses share the following characteristics: (i) they produce non-occluded, enveloped, rod-shaped virions that measure 500–1,000 nm in length and 50–100 nm in diameter; (ii) they possess a large circular double-stranded DNA (dsDNA) genome ranging in size from 120 to 190 kbp and having G + C ratios ranging from 28 to 44%; (iii) they cause overt salivary gland hypertrophy (SGH) symptoms in dipteran adults and partial to complete sterility. The available information on the complete genome sequence of GpSGHV and MdSGHV indicates significant co-linearity between the two viral genomes, whereas no co-linearity was observed with baculoviruses, ascoviruses, entomopoxviruses, iridoviruses and nudiviruses, other large invertebrate DNA viruses. The DNA polymerases encoded by the SGHVs are of the type B and closely related, but they are phylogenetically distant from DNA polymerases encoded by other large dsDNA viruses. The great majority of SGHV ORFs could not be assigned by sequence comparison. Phylogenetic analysis of conserved genes clustered both SGHVs, but distantly from the nudiviruses and baculoviruses. On the basis of the available morphological, (patho)biological, genomic and phylogenetic data, we propose that the two viruses are members of a new virus family named Hytrosaviridae. This proposed family currently comprises two unassigned species, G. pallidipes salivary gland hypertrophy virus and M. domestica salivary gland hypertrophy virus, and a tentative unassigned species, M. equestris salivary gland hypertrophy virus. Here, we present the characteristics and the justification for establishing this new virus family.

The Mediterranean fruit fly (medfly), Ceratitis capitata, is a pest of worldwide substantial economic importance, as well as a Tephritidae model for sterile insect technique (SIT) applications. The latter is partially due to the development and utilization of genetic sexing strains (GSS) for this species, such as the Vienna 8 strain, which is currently used in mass rearing facilities worldwide. Improving the performance of such a strain both in mass rearing facilities and in the field could significantly enhance the efficacy of SIT and reduce operational costs. Recent studies have suggested that the manipulation of gut symbionts can have a significant positive effect on the overall fitness of insect strains. We used culture-based approaches to isolate and characterize gut-associated bacterial species of the Vienna 8 strain under mass rearing conditions. We also exploited one of the isolated bacterial species, Enterobacter sp., as dietary supplement (probiotic) to the larval diet, and we assessed its effects on fitness parameters under the standard operating procedures used in SIT operational programs. Probiotic application of Enterobacter sp. resulted in improvement of both pupal and adult productivity, as well as reduced rearing duration, particularly for males, without affecting pupal weight, sex ratio, male mating competitiveness, flight ability and longevity under starvation.