Investigation of Developmental Stage/Age, Gamma Irradiation Dose, and Temperature in Sterilization of Male Aedes aegypti (Diptera: Culicidae) in a Sterile Insect Technique Program

The sterilization process using gamma irradiation is a crucial component in a program using sterile insect technique (SIT) to control Aedes aegypti. Unfortunately, there is no efficient standard protocol for sterilizing mosquitoes that can produce a high level of sterility while maintaining mating ability and longevity. Therefore, we conducted a study of the critical factors necessary to develop such a standard protocol. In this study, male Ae. aegypti pupae, as well as adults aged 1 d and 3 d, were irradiated using a Gamma-cell 220 irradiator doses of 0, 20, 40, 60, 70, 80, and 100 Gray (Gy). In addition, male Ae. aegypti in the pupal and adult stage aged 1 d were irradiated at a dose of 70 Gy at various temperatures. Changes in emergence rates, longevity, sterility, and mating competitiveness were recorded for each combination of parameters. Results showed that an increase of irradiation dose leads to a rise of induced sterility at all developmental stages, while simultaneously reducing emergence rate, survival, and mating competitiveness. Higher temperatures resulted in increased levels of sterility, reduced longevity, and did not affect the ability to mate. This study found that an irradiation dose of 70 Gy at a temperature between 20.00 and 22.30°C administered in the pupal stage induced a high level of sterility (around 98%), while maintaining mating competitiveness and longevity.

Genetic structure and symbiotic profile of worldwide natural populations of the Mediterranean fruit fly, Ceratitis capitata

Background The Mediterranean fruit fly, Ceratitis capitata, is a cosmopolitan agricultural pest of worldwide economic importance and a model for the development of the Sterile Insect Technique (SIT) for fruit flies of the Tephritidae family (Diptera). SIT relies on the effective mating of laboratory-reared strains and natural populations, and therefore requires an efficient mass-rearing system that will allow for the production of high-quality males. Adaptation of wild flies to an artificial laboratory environment can be accompanied by negative effects on several life history traits through changes in their genetic diversity and symbiotic communities. Such changes may lead to reduced biological quality and mating competitiveness in respect to the wild populations. Profiling wild populations can help understand, and maybe reverse, deleterious effects accompanying laboratory domestication thus providing insects that can efficiently and effectively support SIT application. Results In the present study, we analyzed both the genetic structure and gut symbiotic communities of natural medfly populations of worldwide distribution, including Europe, Africa, Australia, and the Americas. The genetic structure of 408 individuals from 15 distinct populations was analyzed with a set of commonly used microsatellite markers. The symbiotic communities of a subset of 265 individuals from 11 populations were analyzed using the 16S rRNA gene-based amplicon sequencing of single individuals (adults). Genetic differentiation was detected among geographically distant populations while adults originated from neighboring areas were genetically closer. Alpha and beta diversity of bacterial communities pointed to an overall reduced symbiotic diversity and the influence of the geographic location on the bacterial profile. Conclusions Our analysis revealed differences both in the genetic profile and the structure of gut symbiotic communities of medfly natural populations. The genetic analysis expanded our knowledge to populations not analyzed before and our results were in accordance with the existing scenarios regarding this species expansion and colonization pathways. At the same time, the bacterial communities from different natural medfly populations have been characterized, thus broadening our knowledge on the microbiota of the species across its range. Genetic and symbiotic differences between natural and laboratory populations must be considered when designing AW-IPM approaches with a SIT component, since they may impact mating compatibility and mating competitiveness of the laboratory-reared males. In parallel, enrichment from wild populations and/or symbiotic supplementation could increase rearing productivity, biological quality, and mating competitiveness of SIT-important laboratory strains.

Fluorescent markers rhodamine B and uranine for Anopheles gambiae adults and matings

Background Marking mosquitoes is vital for mark-release-recapture and many laboratory studies, but their small size precludes the use of methods that are available for larger animals such as unique identifier tags and radio devices. Fluorescent dust is the most commonly used method to distinguish released individuals from the wild population. Numerous colours and combinations can be used, however, dust sometimes affects longevity and behaviour so alternatives that do not have these effects would contribute substantially. Rhodamine B has previously been demonstrated to be useful for marking adult Aedes aegypti males when added to the sugar meal. Unlike dust, this also marked the seminal fluid making it possible to detect matings by marked males in the spermatheca of females. Here, marking of Anopheles gambiae sensu stricto with rhodamine B and uranine was performed to estimate their potential contribution. Methods Two fluorescent markers, rhodamine B and uranine, were dissolved in sugar water and fed to adult An. gambiae. Concentrations that are useful for marking individuals and seminal fluid were determined. The effects on adult longevity, the durability of the marking and detection of the marker in mated females was determined. Male mating competitiveness was also evaluated.Results Rhodamine B marking in adults is detectable for at least three weeks, however uranine marking declines with time and at low doses can be confused with auto-fluorescence. Both can be used for marking seminal fluid which can be detected in females mated by marked males, but, again, at low concentrations uranine-marking is more easily confused with the natural fluorescence of seminal fluid. Neither dye affected mating competitiveness.Conclusions Both markers tested could be useful for field and laboratory studies. Their use has substantial potential to contribute to a greater understanding of the bio-ecology of this important malaria vector. Rhodamine B has the advantage that it appears to be permanent and is less easily confused with auto-fluorescence. The primary limitation of both methods is that sugar feeding is necessary for marking and adults must be held for at least 2 nights to ensure all individuals are marked whereas dusts provide immediate and thorough marking.

Fluorescent markers rhodamine B and uranine for Anopheles gambiae adults and matings

Background Marking mosquitoes is vital for mark-release-recapture and many laboratory studies, but their small size precludes the use of methods that are available for larger animals such as unique identifier tags and radio devices. Fluorescent dust is the most commonly used method to distinguish released individuals from the wild population. Numerous colors and combinations can be used, however, dust sometimes affects longevity and behavior so alternatives that do not have these effects would contribute substantially. Rhodamine B has previously been demonstrated to be useful for marking adult Aedes aegypti males when added to the sugar meal. Unlike dust, this also marked the seminal fluid making it possible to detect matings by marked males in the spermatheca of females. Here we evaluate marking in Anopheles gambiae s. s. with rhodamine B and uranine to estimate their potential contribution. Methods Two fluorescent markers, rhodamine B and uranine, were dissolved in sugar water and fed to adult Anopheles gambiae. Concentrations that are useful for marking individuals and seminal fluid were determined. The effects on adult longevity, the durability of the marking and detection of the marker in mated females was determined. We also evaluated effects on mating competitiveness. Results Rhodamine B marking in adults is detectable for at least three weeks, however uranine marking declines with time and at low doses can be confused with auto-fluorescence. Both can be used for marking seminal fluid which can be detected in females mated by marked males, but, again, at low concentrations uranine-marking is more easily confused with the natural fluorescence of seminal fluid. Neither dye affected mating competitiveness. Conclusions Both markers tested could be useful for field and laboratory studies. Their use has substantial potential to contribute to a greater understanding of the bio-ecology of this important malaria vector. Rhodamine B has the advantage that it appears to be permanent and is less easily confused with auto-fluorescence. The primary limitation of both methods is that sugar feeding is necessary for marking and adults must be held for at least 2 nights to ensure all individuals are marked whereas dusts provide immediate and thorough marking.

A new automated chilled adult release system for the aerial distribution of sterile male tsetse flies

Background: Tsetse flies transmit trypanosomes that cause the debilitating diseases human African trypanosomosis (HAT) or sleeping sickness in humans and animal African trypanosomosis (AAT) or nagana in livestock. The riverine tsetse species Glossina palpalis gambiensis Vanderplank (Diptera: Glossinidae) inhabits riparian forests along river systems in West Africa. The Government of Senegal has embarked on a project to eliminate a population of this tsetse species from the Niayes area with the objective to manage AAT in the area. The project is implemented following an area-wide integrated pest management approach with an SIT component. The SIT can only be successful when the sterile males that are released in the field are of high biological quality, i.e. have the same dispersal capacity, survival and competitiveness as their wild counterparts. To date, sterile tsetse males have been released by air using biodegradable cardboard cartons that were manually dropped from a fixed-wing aircraft or gyrocopter. The cardboard boxes are however expensive, and the system is rather cumbersome to implement. Methods: A new prototype of an automated chilled adult release system (Bruno Spreader Innovation, (BSI™)) for tsetse flies was tested for its accuracy (in counting numbers of sterile males as loaded into the machine), release rate consistency and impact on quality of the released males. The impact of the release process was evaluated on several performance indicators of the irradiated male flies such as flight propensity, survival, mating competitiveness, premating and mating duration, and insemination rate of mated females. Results: The BSI TM release system counted with a consistent accuracy and released homogenously tsetse flies at the lowest motor speed (0.6 rpm). In addition, the chilling conditions (6 ± 1 o C) and the release process (passing of flies through the machine) had no significant negative impact on the males' flight propensity. No significant differences were observed between the control males (no irradiation and no exposure to the release process), irradiated males (no exposure to the release process) and irradiated males exposed to the release process with respect to mating competitiveness, premating period and mating duration. Only survival of irradiated males that were exposed to the release process was reduced, irrespective of whether the males were held with or without feeding. Conclusion: Although the release process had a negative effect on survival of the flies, the data of the experiments indicate that the BSI machine holds promise for use in operational tsetse SIT programmes. The promising results of this study will now need to be confirmed under operational field conditions in West Africa.