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Reproductive sterility is the basis of the sterile insect technique (SIT) and essential for its success in the field. Numerous factors that influence dose–response in insects have been identified. However, historically the radiation dose administered has been considered a constant. Efforts aiming to standardize protocols for mosquito irradiation found that, despite carefully controlling many variable factors, there was still an unknown element responsible for differences in expected sterility levels of insects irradiated with the same dose and handling protocols. Thus, together with previous inconclusive investigations, the question arose whether dose really equals dose in terms of biological response, no matter the rate at which the dose is administered. Interestingly, the dose rate effects studied in human nuclear medicine indicated that dose rate could alter dose–response in mammalian cells. Here, we conducted experiments to better understand the interaction of dose and dose rate to assess the effects in irradiated mosquitoes. Our findings suggest that not only does dose rate alter irradiation-induced effects, but that the interaction is not linear and may change with dose. We speculate that the recombination of reactive oxygen species (ROS) in treatments with moderate to high dose rates might minimize indirect radiation-induced effects in mosquitoes and decrease sterility levels, unless dose along with its direct effects is increased. Together with further studies to identify an optimum match of dose and dose rate, these results could assist in the development of improved methods for the production of high-quality sterile mosquitoes to enhance the efficiency of SIT programs.

Tsetse eradication continues to be a top priority for African governments including that of Senegal, which embarked on a project to eliminate Glossina palpalis gambiensis from the Niayes area, following an area-wide integrated pest management approach with an SIT component. A successful SIT programme requires competitive sterile males of high biological quality. This may be hampered by handling processes including irradiation and the release mechanisms, necessitating continued improvement of these processes, to maintain the quality of flies. A new prototype of an automated chilled adult release system (Bruno Spreader Innovation, (BSI[TM])) for tsetse flies was tested for its accuracy (in counting) and release rate consistency. Also, its impact on the quality of the released sterile males was evaluated on performance indicators, including flight propensity, mating competitiveness, premating and mating duration, insemination rate of mated females and survival of male flies. The BSI.sup.TM release system accurately counted and homogenously released flies at the lowest motor speed set (0.6 rpm), at a consistent rate of 60±9.58 males/min. Also, the release process, chilling (6 ± 1°C) and passing of flies through the machine) had no significant negative impact on the male flight propensity, mating competitiveness, premating and mating durations and the insemination rates. Only the survival of flies was negatively affected whether under feeding or starvation. The positive results of this study show that the BSI[TM] release system is promising for use in future tsetse SIT programmes. However, the negative impact of the release process on survival of flies needs to be addressed in future studies and results of this study confirmed under operational field conditions in West Africa.

The Sterile Insect Technique (SIT) is a successful autocidal control method that uses ionizing radiation to sterilize insects. However, irradiation in normal atmospheric conditions can be damaging for males, because irradiation generates substantial biological oxidative stress that, combined with domestication and mass-rearing conditions, may reduce sterile male sexual competitiveness and quality. In this study, biological oxidative stress and antioxidant capacity were experimentally manipulated in Anastrepha suspensa using a combination of low-oxygen conditions and transgenic overexpression of mitochondrial superoxide dismutase (SOD2) to evaluate their role in the sexual behavior and quality of irradiated males. Our results showed that SOD2 overexpression enhances irradiated insect quality and improves male competitiveness in leks. However, the improvements in mating performance were modest, as normoxia-irradiated SOD2 males exhibited only a 22% improvement in mating success compared to normoxia-irradiated wild type males. Additionally, SOD2 overexpression did not synergistically improve the mating success of males irradiated in either hypoxia or severe hypoxia. Short-term hypoxic and severe-hypoxic conditioning hormesis, per se, increased antioxidant capacity and enhanced sexual competitiveness of irradiated males relative to non-irradiated males in leks. Our study provides valuable new information that antioxidant enzymes, particularly SOD2, have potential to improve the quality and lekking performance of sterile males used in SIT programs.

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® r RNA-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.

Background Radiation induced sterility is the basis of the Sterile Insect Technique, by which a target insect pest population is suppressed by releasing artificially reared sterile males of the pest species in overflooding numbers over a target site. In order for the sterile males to be of high biological quality, effective standard irradiation protocols are required. Following studies investigating the effects of mosquito pupae irradiation in water versus in air, there is a need to investigate the oxy-regulatory behavior of mosquito pupae in water to better understand the consequences of irradiation in hypoxic versus normoxic conditions. Methods Pupae of Aedes aegypti , Ae. albopictus , and Anopheles arabiensis were submerged in water inside air-tight 2 ml glass vials at a density of 100 pupae/ml and the dissolved oxygen (DO) levels in the water were measured and plotted over time. In addition, male pupae of Ae. aegypti (aged 40–44 h), Ae. albopictus (aged 40–44 h) and An. arabiensis (aged 20–24 h) were irradiated in a gammacell220 at increasing doses in either hypoxic (water with < 0.5% O 2 content) or normoxic (in air) conditions. The males were then mated to virgin females and resulting eggs were checked for induced sterility. Results All three species depleted the water of DO to levels under 0.5% within 30 minutes, with An. arabiensis consuming oxygen the fastest at under 10 minutes. Following irradiation, the protective effect of hypoxia was observed across species and doses ( P  < 0.0001), increasing at higher doses. This effect was most pronounced in An. arabiensis . Conclusions The consumption of dissolved oxygen by pupae submerged in water was significantly different between species, indicating that their oxy-regulatory capacity seems to have possibly evolved according to their preferred breeding site characteristics. This needs to be considered when sterilizing male mosquitoes at pupal stage in water. Depending on species, their DO consumption rates and their density, irradiation doses needed to achieve full sterility may vary significantly. Further assessments are required to ascertain optimal conditions in terms of ambient atmosphere during pupal irradiation to produce competitive sterile males, and temperature and density dependent effects are expected.

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. 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.

There is currently renewed interest in assessing the feasibility of the sterile insect technique (SIT) to control African malaria vectors in designated areas. The SIT relies on the sterilization of males before mass release, with sterilization currently being achieved through the use of ionizing radiation. This paper reviews previous work on radiation sterilization of Anopheles mosquitoes. In general, the pupal stage was irradiated due to ease of handling compared to the adult stage. The dose-response curve between the induced sterility and log (dose) was shown to be sigmoid, and there was a marked species difference in radiation sensitivity. Mating competitiveness studies have generally been performed under laboratory conditions. The competitiveness of males irradiated at high doses was relatively poor, but with increasing ratios of sterile males, egg hatch could be lowered effectively. Males irradiated as pupae had a lower competitiveness compared to males irradiated as adults, but the use of partially-sterilizing doses has not been studied extensively. Methods to reduce somatic damage during the irradiation process as well as the use of other agents or techniques to induce sterility are discussed. It is concluded that the optimal radiation dose chosen for insects that are to be released during an SIT programme should ensure a balance between induced sterility of males and their field competitiveness, with competitiveness being determined under (semi-) field conditions. Self-contained 60 Co research irradiators remain the most practical irradiators but these are likely to be replaced in the future by a new generation of high output X ray irradiators.

Tsetse flies transmit trypanosomes that cause human and African animal trypanosomosis, a debilitating disease of humans (sleeping sickness) and livestock (nagana). An area-wide integrated pest management campaign against Glossina palpalis gambiensis has been implemented in Senegal since 2010 that includes a sterile insect technique (SIT) component. The SIT can only be successful when the sterile males that are destined for release have a flight ability, survival and competitiveness that are as close as possible to that of their wild male counterparts. Tests were developed to assess the quality of G. p. gambiensis males that emerged from pupae that were produced and irradiated in Burkina Faso and Slovakia (irradiation done in Seibersdorf, Austria) and transported weekly under chilled conditions to Dakar, Senegal. For each consignment a sample of 50 pupae was used for a quality control test (QC group). To assess flight ability, the pupae were put in a cylinder filtering emerged flies that were able to escape the cylinder. The survival of these flyers was thereafter monitored under stress conditions (without feeding). Remaining pupae were emerged and released in the target area of the eradication programme (RF group). The following parameter values were obtained for the QC flies: average emergence rate more than 69%, median survival of 6 days, and average flight ability of more than 35%. The quality protocol was a good proxy of fly quality, explaining a large part of the variances of the examined parameters. The quality protocol described here will allow the accurate monitoring of the quality of shipped sterile male tsetse used in operational eradication programmes in the framework of the Pan-African Tsetse and Trypanosomosis Eradication Campaign.

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.

Background Tsetse control is considered an effective and sustainable tactic for the control of cyclically transmitted trypanosomosis in the absence of effective vaccines and inexpensive, effective drugs. The sterile insect technique (SIT) is currently used to eliminate tsetse fly populations in an area-wide integrated pest management (AW-IPM) context in Senegal. For SIT, tsetse mass rearing is a major milestone that associated microbes can influence. Tsetse flies can be infected with microorganisms, including the primary and obligate Wigglesworthia glossinidia , the commensal Sodalis glossinidius , and Wolbachia pipientis . In addition, tsetse populations often carry a pathogenic DNA virus, the Glossina pallidipes salivary gland hypertrophy virus (GpSGHV) that hinders tsetse fertility and fecundity. Interactions between symbionts and pathogens might affect the performance of the insect host. Methods In the present study, we assessed associations of GpSGHV and tsetse endosymbionts under field conditions to decipher the possible bidirectional interactions in different Glossina species. We determined the co-infection pattern of GpSGHV and Wolbachia in natural tsetse populations. We further analyzed the interaction of both Wolbachia and GpSGHV infections with Sodalis and Wigglesworthia density using qPCR. Results The results indicated that the co-infection of GpSGHV and Wolbachia was most prevalent in Glossina austeni and Glossina morsitans morsitans , with an explicit significant negative correlation between GpSGHV and Wigglesworthia density. GpSGHV infection levels > 10 3.31 seem to be absent when Wolbachia infection is present at high density (> 10 7.36 ), suggesting a potential protective role of Wolbachia against GpSGHV. Conclusion The result indicates that Wolbachia infection might interact (with an undefined mechanism) antagonistically with SGHV infection protecting tsetse fly against GpSGHV, and the interactions between the tsetse host and its associated microbes are dynamic and likely species specific; significant differences may exist between laboratory and field conditions. Graphical Abstract