A Molecular Method to Discriminate between Mass-Reared Sterile and Wild Tsetse Flies during Eradication Programmes That Have a Sterile Insect Technique Component

Background

The Government of Senegal has embarked several years ago on a project that aims to eradicate Glossina palpalis gambiensis from the Niayes area. The removal of the animal trypanosomosis would allow the development more efficient livestock production systems. The project was implemented using an area-wide integrated pest management strategy including a sterile insect technique (SIT) component. The released sterile male flies originated from a colony from Burkina Faso.

Methodology/Principal Findings

Monitoring the efficacy of the sterile male releases requires the discrimination between wild and sterile male G. p. gambiensis that are sampled in monitoring traps. Before being released, sterile male flies were marked with a fluorescent dye powder. The marking was however not infallible with some sterile flies only slightly marked or some wild flies contaminated with a few dye particles in the monitoring traps. Trapped flies can also be damaged due to predation by ants, making it difficult to discriminate between wild and sterile males using a fluorescence camera and / or a fluorescence microscope. We developed a molecular technique based on the determination of cytochrome oxidase haplotypes of G. p. gambiensis to discriminate between wild and sterile males. DNA was isolated from the head of flies and a portion of the 5’ end of the mitochondrial gene cytochrome oxidase I was amplified to be finally sequenced. Our results indicated that all the sterile males from the Burkina Faso colony displayed the same haplotype and systematically differed from wild male flies trapped in Senegal and Burkina Faso. This allowed 100% discrimination between sterile and wild male G. p. gambiensis.

Conclusions/Significance

This tool might be useful for other tsetse control campaigns with a SIT component in the framework of the Pan-African Tsetse and Trypanosomosis Eradication Campaign (PATTEC) and, more generally, for other vector or insect pest control programs.     

Combining paratransgenesis with SIT: impact of ionizing radiation on the DNA copy number of <Emphasis Type="Italic">Sodalis glossinidius in tsetse flies</Emphasis>

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.

     

The Smart Aerial Release Machine,a Universal System for Applying the Sterile Insect Technique

Background

Beyond insecticides, alternative methods to control insect pests for agriculture and vectors of diseases are needed. Management strategies involving the mass-release of living control agents have been developed, including genetic control with sterile insects and biological control with parasitoids, for which aerial release of insects is often required. Aerial release in genetic control programmes often involves the use of chilled sterile insects, which can improve dispersal, survival and competitiveness of sterile males. Currently available means of aerially releasing chilled fruit flies are however insufficiently precise to ensure homogeneous distribution at low release rates and no device is available for tsetse.

Methodology/Principal Findings

Here we present the smart aerial release machine, a new design by the Mubarqui Company, based on the use of vibrating conveyors. The machine is controlled through Bluetooth by a tablet with Android Operating System including a completely automatic guidance and navigation system (MaxNav software). The tablet is also connected to an online relational database facilitating the preparation of flight schedules and automatic storage of flight reports. The new machine was compared with a conveyor release machine in Mexico using two fruit flies species (Anastrepha ludens and Ceratitis capitata) and we obtained better dispersal homogeneity (% of positive traps, p<0.001) for both species and better recapture rates for Anastrepha ludens (p<0.001), especially at low release densities (<1500 per ha). We also demonstrated that the machine can replace paper boxes for aerial release of tsetse in Senegal.

Conclusions/Significance

This technology limits damages to insects and allows a large range of release rates from 10 flies/km² for tsetse flies up to 600 000 flies/km² for fruit flies. The potential of this machine to release other species like mosquitoes is discussed. Plans and operating of the machine are provided to allow its use worldwide.     

Contrasting population structures of two vectors of African trypanosomoses in Burkina Faso: consequences for control

Background

African animal trypanosomosis is a major obstacle to the development of more efficient and sustainable livestock production systems in West Africa. Riverine tsetse species such as Glossina palpalis gambiensis Vanderplank and Glossina tachinoides Westwood are the major vectors. A wide variety of control tactics is available to manage these vectors, but their removal will in most cases only be sustainable if the control effort is targeting an entire tsetse population within a circumscribed area.

Methodology/Principal Findings

In the present study, genetic variation at microsatellite DNA loci was used to examine the population structure of G. p. gambiensis and G. tachinoides inhabiting four adjacent river basins in Burkina Faso, i.e. the Mouhoun, the Comoé, the Niger and the Sissili River Basins. Isolation by distance was significant for both species across river basins, and dispersal of G. tachinoides was ∼3 times higher than that of G. p. gambiensis. Thus, the data presented indicate that no strong barriers to gene flow exists between riverine tsetse populations in adjacent river basins, especially so for G. tachinoides.

Conclusions/Significance

Therefore, potential re-invasion of flies from adjacent river basins will have to be prevented by establishing buffer zones between the Mouhoun and the other river basin(s), in the framework of the PATTEC (Pan African Tsetse and Trypanosomosis Eradication Campaign) eradication project that is presently targeting the northern part of the Mouhoun River Basin. We argue that these genetic analyses should always be part of the baseline data collection before any tsetse control project is initiated.     

Description of a Nanobody-based Competitive Immunoassay to Detect Tsetse Fly Exposure

Background

Tsetse flies are the main vectors of human and animal African trypanosomes. The Tsal proteins in tsetse fly saliva were previously identified as suitable biomarkers of bite exposure. A new competitive assay was conceived based on nanobody (Nb) technology to ameliorate the detection of anti-Tsal antibodies in mammalian hosts.

Methodology/Principal Findings

A camelid-derived Nb library was generated against the Glossina morsitans morsitans sialome and exploited to select Tsal specific Nbs. One of the three identified Nb families (family III, TsalNb-05 and TsalNb-11) was found suitable for anti-Tsal antibody detection in a competitive ELISA format. The competitive ELISA was able to detect exposure to a broad range of tsetse species (G. morsitans morsitans, G. pallidipes, G. palpalis gambiensis and G. fuscipes) and did not cross-react with the other hematophagous insects (Stomoxys calcitrans and Tabanus yao). Using a collection of plasmas from tsetse-exposed pigs, the new test characteristics were compared with those of the previously described G. m. moristans and rTsal1 indirect ELISAs, revealing equally good specificities (> 95%) and positive predictive values (> 98%) but higher negative predictive values and hence increased sensitivity (> 95%) and accuracy (> 95%).

Conclusion/Significance

We have developed a highly accurate Nb-based competitive immunoassay to detect specific anti-Tsal antibodies induced by various tsetse fly species in a range of hosts. We propose that this competitive assay provides a simple serological indicator of tsetse fly presence without the requirement of test adaptation to the vertebrate host species. In addition, the use of monoclonal Nbs for antibody detection is innovative and could be applied to other tsetse fly salivary biomarkers in order to achieve a multi-target immunoprofiling of hosts. In addition, this approach could be broadened to other pathogenic organisms for which accurate serological diagnosis remains a bottleneck.     

Towards an optimal design of target for tsetse control: comparisons of novel targets for the control of Palpalis group tsetse in West Africa

Background

Tsetse flies of the Palpalis group are the main vectors of sleeping sickness in Africa. Insecticide impregnated targets are one of the most effective tools for control. However, the cost of these devices still represents a constraint to their wider use. The objective was therefore to improve the cost effectiveness of currently used devices.

Methodology/Principal Findings

Experiments were performed on three tsetse species, namely Glossina palpalis gambiensis and G. tachinoides in Burkina Faso and G. p. palpalis in Côte d''Ivoire. The 1×1 m² black blue black target commonly used in W. Africa was used as the standard, and effects of changes in target size, shape, and the use of netting instead of black cloth were measured. Regarding overall target shape, we observed that horizontal targets (i.e. wider than they were high) killed 1.6-5x more G. p. gambiensis and G. tachinoides than vertical ones (i.e. higher than they were wide) (P<0.001). For the three tsetse species including G. p. palpalis, catches were highly correlated with the size of the target. However, beyond the size of 0.75 m, there was no increase in catches. Replacing the black cloth of the target by netting was the most cost efficient for all three species.

Conclusion/Significance

Reducing the size of the current 1*1 m black-blue-black target to horizontal designs of around 50 cm and replacing black cloth by netting will improve cost effectiveness six-fold for both G. p. gambiensis and G. tachinoides. Studying the visual responses of tsetse to different designs of target has allowed us to design more cost-effective devices for the effective control of sleeping sickness and animal trypanosomiasis in Africa.     

Impact of <Emphasis Type="Italic">Glossina pallidipes</Emphasis> salivary gland hypertrophy virus (GpSGHV) on a heterologous tsetse fly host, <Emphasis Type="Italic">Glossina fuscipes fuscipes</Emphasis>

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.

     

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

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.

     

Male Sexual Behavior and Pheromone Emission Is Enhanced by Exposure to Guava Fruit Volatiles in Anastrepha fraterculus

Background

Plant chemicals can affect reproductive strategies of tephritid fruit flies by influencing sex pheromone communication and increasing male mating competitiveness.

Objective and Methodology

We explored whether exposure of Anastrepha fraterculus males to guava fruit volatiles and to a synthetic blend of volatile compounds released by this fruit affects the sexual performance of wild and laboratory flies. By means of bioassays and pheromone collection we investigated the mechanism underlying this phenomenon.

Results

Guava volatile exposure enhanced male mating success and positively affected male calling behavior and pheromone release in laboratory and wild males. Changes in male behavior appear to be particularly important during the initial phase of the sexual activity period, when most of the mating pairs are formed. Exposure of laboratory males to a subset of guava fruit volatiles enhanced mating success, showing that the response to the fruit might be mimicked artificially.

Conclusions

Volatiles of guava seem to influence male mating success through an enhancement of chemical and physical signals related to the communication between sexes. This finding has important implications for the management of this pest species through the Sterile Insect Technique. We discuss the possibility of using artificial blends to improve the sexual competitiveness of sterile males.     

The interaction of dispersal and control methods for the riverine tsetse fly Glossina palpalis gambiensis (Diptera: Glossinidae): a modelling study

We used a spatial model of a riverine tsetse fly species Glossina palpalis gambiensis life cycle to investigate the interaction between their dispersal and three control methods and to document these interactions using sensitivity analyses. The model is currently limited to gallery forest habitat inhabited by Glossina palpalis gambiensis in the dry season in the sub-humid zone of West Africa. The control methods modelled were traps and targets (TT), insecticide-treated livestock (ITL), and the sterile insect technique (SIT). Both distance dispersed (up to 800 m) and percent of flies dispersing each day (up to 60 %) increased the efficiency of control by TT. Most of this increase occurred for low values of both distance dispersed and percent dispersing, but the increase continued up to the limits tried. The daily movement of cattle assisted the control program and when movement was considerable (up to 600 m daily) the effects were greater than the effects of tsetse dispersal. Random dispersal decreased aggregation and equilibrium population size, and thus also increased the efficiency of SIT. Dispersal that was mostly oriented towards clumps was of much less value for SIT but acted on TT and ITL similarly to random dispersal.     

Prevalence of trypanosomes,salivary gland hypertrophy virus and <Emphasis Type="Italic">Wolbachia</Emphasis> 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.

     

Mycobacterium tuberculosis Genotypes Determined by Spoligotyping to Be Circulating in Colombia between 1999 and 2012 and Their Possible Associations with Transmission and Susceptibility to First-Line Drugs

Introduction

Tuberculosis (TB) remains a primary public health problem worldwide. The number of multidrug-resistant tuberculosis (MDR TB) cases has increased in recent years in Colombia. Knowledge of M. tuberculosis genotypes defined by spoligotyping can help determine the circulation of genotypes that must be controlled to prevent the spread of TB.

Objective

To describe the genotypes of M. tuberculosis using spoligotyping in resistant and drug-sensitive isolates and their possible associations with susceptibility to first-line drugs.

Methods

An analytical observational study was conducted that included 741 isolates of M. tuberculosis from patients. The isolates originated from 31 departments and were obtained by systematic surveillance between 1999 and 2012.

Results

In total 61.94% of the isolates were resistant to 1 or more drugs, and 147 isolates were MDR. In total, 170 genotypes were found in the population structure of Colombian M. tuberculosis isolates. The isolates were mainly represented by four families: LAM (39.9%), Haarlem (19%), Orphan (17%) and T (9%). The SIT42 (LAM 9) was the most common genotype and contained 24.7% of the isolates, followed by the genotypes SIT62 (Haarlem1), SIT53 (T1), and SIT50 (H3). A high clustering of isolates was evident with 79.8% of the isolates classified into 32 groups. The Beijing family was associated with resistant isolates, whereas the Haarlem and T families were associated with sensitive isolates. The Haarlem family was also associated with grouped isolates (p = 0.031).

Conclusions

A high proportion (approximately 80%) of isolates was found in clusters; these clusters were not associated with resistance to first-line drugs. The Beijing family was associated with drug resistance, whereas the T and Haarlem families were associated with susceptibility in the Colombian isolates studied.     

Illuminating the Prevalence of Trypanosoma brucei s.l. in Glossina Using LAMP as a Tool for Xenomonitoring

Background

As the reality of eliminating human African trypanosomiasis (HAT) by 2020 draws closer, the need to detect and identify the remaining areas of transmission increases. Here, we have explored the feasibility of using commercially available LAMP kits, designed to detect the Trypanozoon group of trypanosomes, as a xenomonitoring tool to screen tsetse flies for trypanosomes to be used in future epidemiological surveys.

Methods and Findings

The DNA extraction method was simplified and worked with the LAMP kits to detect a single positive fly when pooled with 19 negative flies, and the absolute lowest limit of detection that the kits were able to work at was the equivalent of 0.1 trypanosome per ml. The DNA from Trypanosoma brucei brucei could be detected six days after the fly had taken a blood meal containing dead trypanosomes, and when confronted with a range of non-target species, from both laboratory-reared flies and wild-caught flies, the kits showed no evidence of cross-reacting.

Conclusion

We have shown that it is possible to use a simplified DNA extraction method in conjunction with the pooling of tsetse flies to decrease the time it would take to screen large numbers of flies for the presence of Trypanozoon trypanosomes. The use of commercially-available LAMP kits provides a reliable and highly sensitive tool for xenomonitoring and identifying potential sleeping sickness transmission sites.     

Ex-ante Benefit-Cost Analysis of the Elimination of a Glossina palpalis gambiensis Population in the Niayes of Senegal

Background

In 2005, the Government of Senegal embarked on a campaign to eliminate a Glossina palpalis gambiensis population from the Niayes area (∼1000 km²) under the umbrella of the Pan African Tsetse and Trypanosomosis Eradication Campaign (PATTEC). The project was considered an ecologically sound approach to intensify cattle production. The elimination strategy includes a suppression phase using insecticide impregnated targets and cattle, and an elimination phase using the sterile insect technique, necessary to eliminate tsetse in this area.

Methodology/Principal Findings

Three main cattle farming systems were identified: a traditional system using trypanotolerant cattle and two “improved” systems using more productive cattle breeds focusing on milk and meat production. In improved farming systems herd size was 45% lower and annual cattle sales were €250 (s.d. 513) per head as compared to €74 (s.d. 38) per head in traditional farming systems (p<10⁻³). Tsetse distribution significantly impacted the occurrence of these farming systems (p = 0.001), with 34% (s.d. 4%) and 6% (s.d. 4%) of improved systems in the tsetse-free and tsetse-infested areas, respectively. We calculated the potential increases of cattle sales as a result of tsetse elimination considering two scenarios, i.e. a conservative scenario with a 2% annual replacement rate from traditional to improved systems after elimination, and a more realistic scenario with an increased replacement rate of 10% five years after elimination. The final annual increase of cattle sales was estimated at ∼€2800/km² for a total cost of the elimination campaign reaching ∼€6400/km².

Conclusion/Significance

Despite its high cost, the benefit-cost analysis indicated that the project was highly cost-effective, with Internal Rates of Return (IRR) of 9.8% and 19.1% and payback periods of 18 and 13 years for the two scenarios, respectively. In addition to an increase in farmers'' income, the benefits of tsetse elimination include a reduction of grazing pressure on the ecosystems.     

A Spatio-temporal Model of African Animal Trypanosomosis Risk

Background

African animal trypanosomosis (AAT) is a major constraint to sustainable development of cattle farming in sub-Saharan Africa. The habitat of the tsetse fly vector is increasingly fragmented owing to demographic pressure and shifts in climate, which leads to heterogeneous risk of cyclical transmission both in space and time. In Burkina Faso and Ghana, the most important vectors are riverine species, namely Glossina palpalis gambiensis and G. tachinoides, which are more resilient to human-induced changes than the savannah and forest species. Although many authors studied the distribution of AAT risk both in space and time, spatio-temporal models allowing predictions of it are lacking.

Methodology/Principal Findings

We used datasets generated by various projects, including two baseline surveys conducted in Burkina Faso and Ghana within PATTEC (Pan African Tsetse and Trypanosomosis Eradication Campaign) national initiatives. We computed the entomological inoculation rate (EIR) or tsetse challenge using a range of environmental data. The tsetse apparent density and their infection rate were separately estimated and subsequently combined to derive the EIR using a “one layer-one model” approach. The estimated EIR was then projected into suitable habitat. This risk index was finally validated against data on bovine trypanosomosis. It allowed a good prediction of the parasitological status (r² = 67%), showed a positive correlation but less predictive power with serological status (r² = 22%) aggregated at the village level but was not related to the illness status (r² = 2%).

Conclusions/Significance

The presented spatio-temporal model provides a fine-scale picture of the dynamics of AAT risk in sub-humid areas of West Africa. The estimated EIR was high in the proximity of rivers during the dry season and more widespread during the rainy season. The present analysis is a first step in a broader framework for an efficient risk management of climate-sensitive vector-borne diseases.     

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.

     

Raspberry ketone-supplemented diet has no effect on fitness parameters or lure responsiveness in male melon flies,Zeugodacus cucurbitae (Diptera: Tephritidae)

The male annihilation technique (MAT) and sterile insect technique (SIT) are often used to control pestiferous tephritid fruit flies (Diptera: Tephritidae). MAT involves the deployment of traps containing a male attractant and insecticide with the goal of drastically reducing male abundance and ultimately eliminating the entire population. SIT, which involves the mass production, sterilization, and release of the target species, may also be implemented to achieve final extirpation. Generally, simultaneous implementation of MAT and SIT is counterproductive, because the presence of large numbers of male-specific traps in the environment (MAT) would greatly reduce the number of sterile males available for copulating with wild females (SIT). However, studies on the Queensland fruit fly, Bactrocera tryoni (Froggatt), indicate that concurrent use of MAT and SIT may be feasible. Sexually mature males of B. tryoni are attracted to the raspberry ketone and its synthetic analogue cue-lure. Males of B. tryoni fed raspberry-ketone-supplemented diet when newly emerged showed lower attraction to cue-lure baited traps than control males. In addition, newly emerged males provided this diet displayed accelerated sexual maturation, which would allow the early release of sterile males and reduce pre-release holding costs. Here, we examined whether the addition of raspberry ketone to the adult diet of male melon flies, Zeugodacus cucurbitae (Coquillett), produced effects similar to those observed for B. tryoni. Despite using similar methods, no significant effect of raspberry ketone-supplemented diet on time to sexual maturity, survival, mating competitiveness, or attraction to cue-lure baited traps in mass-reared Z. cucurbitae males.     

Identification of Algerian Field-Caught Phlebotomine Sand Fly Vectors by MALDI-TOF MS

Background

Phlebotomine sand flies are known to transmit Leishmania parasites, bacteria and viruses that affect humans and animals in many countries worldwide. Precise sand fly identification is essential to prevent phlebotomine-borne diseases. Over the past two decades, progress in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has emerged as an accurate tool for arthropod identification. The objective of the present study was to investigate the usefulness of MALDI-TOF MS as a tool for identifying field-caught phlebotomine.

Methodology/Principal Findings

Sand flies were captured in four sites in north Algeria. A subset was morphologically and genetically identified. Six species were found in these areas and a total of 28 stored frozen specimens were used for the creation of the reference spectrum database. The relevance of this original method for sand fly identification was validated by two successive blind tests including the morphological identification of 80 new specimens which were stored at -80°C, and 292 unknown specimens, including engorged specimens, which were preserved under different conditions. Intra-species reproducibility and inter-species specificity of the protein profiles were obtained, allowing us to distinguish specimens at the gender level. Querying of the sand fly database using the MS spectra from the blind test groups revealed concordant results between morphological and MALDI-TOF MS identification. However, MS identification results were less efficient for specimens which were engorged or stored in alcohol. Identification of 362 phlebotomine sand flies, captured at four Algerian sites, by MALDI-TOF MS, revealed that the subgenus Larroussius was predominant at all the study sites, except for in M’sila where P. (Phlebotomus) papatasi was the only sand fly species detected.

Conclusion

The present study highlights the application of MALDI-TOF MS for monitoring sand fly fauna captured in the field. The low cost, reliability and rapidity of MALDI-TOF MS analyses opens up new ways in the management of phlebotomine sand fly-borne diseases.     

Variation in Pollen-Donor Composition among Pollinators in an Entomophilous Tree Species,Castanea crenata,Revealed by Single-Pollen Genotyping

Background

In plants, reproductive success is largely determined by the composition of pollen (i.e., self-pollen and outcross-pollen from near and distant pollen-donors) transported as a result of pollinator foraging behavior (e.g., pollen carryover). However, little evidence is available on how and to what extent the pollen carryover affects the pollen-donor composition and on which insect taxa are effective outcross-pollen transporters under field conditions. In this study, we explored roles of foraging behavior of insect pollinators on pollen-donor composition and subsequent reproductive success in a woody plant.

Methods

We performed paternity analyses based on microsatellite genotyping of individual pollen grains found on diurnal pollinators (i.e., bumblebee, small bee, fly, small beetle, and honeybee) visiting Castanea crenata trees.

Results

The outcross-pollen rate was highest in bumblebees (66%), followed by small bees (35%), flies (31%), and small beetles (18%). The effective number of pollen donors, representing pollen carryover, was greater in bumblebees (9.71) than in flies (3.40), small bees (3.32), and small beetles (3.06). The high percentages of pollen from outside the plot on bumblebees (65.4%) and flies (71.2%) compared to small bees (35.3%) and small beetles (13.5%) demonstrated their longer pollen dispersal distances.

Conclusions

All of the diurnal insects carried outcross-pollen grains for long distances via pollen carryover. This fact suggests that a wide range of insect taxa are potential outcross-pollen transporters for the self-incompatible C. crenata.     

Development,genetic and cytogenetic analyses of genetic sexing strains of the Mexican fruit fly, <Emphasis Type="Italic">Anastrepha ludens</Emphasis> Loew (Diptera: Tephritidae)

Background

Anastrepha ludens is among the pests that have a major impact on México's economy because it attacks fruits as citrus and mangoes. The Mexican Federal government uses integrated pest management to control A. ludens through the Programa Nacional Moscas de la Fruta [National Fruit Fly Program, SAGARPA-SENASICA]. One of the main components of this program is the sterile insect technique (SIT), which is used to control field populations of the pest by releasing sterile flies.

Results

To increase the efficiency of this technique, we have developed a genetic sexing strain (GSS) in which the sexing mechanism is based on a pupal colour dimorphism (brown-black) and is the result of a reciprocal translocation between the Y chromosome and the autosome bearing the black pupae (bp) locus. Ten strains producing wild-type (brown pupae) males and mutant (black pupae) females were isolated. Subsequent evaluations for several generations were performed in most of these strains. The translocation strain named Tapachula-7 showed minimal effect on survival and the best genetic stability of all ten strains. Genetic and cytogenetic analyses were performed using mitotic and polytene chromosomes and we succeeded to characterize the chromosomal structure of this reciprocal translocation and map the autosome breakpoint, despite the fact that the Y chromosome is not visible in polytene nuclei following standard staining.

Conclusions

We show that mitotic and polytene chromosomes can be used in cytogenetic analyses towards the development of genetic control methods in this pest species. The present work is the first report of the construction of GSS of Anastrepha ludens, with potential use in a future Moscafrut operational program.