Benchmarking vector arthropod culture: an example using the African malaria mosquito, Anopheles gambiae (Diptera: Culicidae)

BACKGROUND: Numerous important characteristics of adult arthropods are related to their size; this is influenced by conditions experienced as immatures. Arthropods cultured in the laboratory for research, or mass-reared for novel control methods, must therefore be of a standard size range and known quality so that results are reproducible. METHODS: A simple two-step technique to assess laboratory culture methods was demonstrated using the mosquito Anopheles gambiae s.s. as a model. First, the ranges of key development outcomes were determined using various diet levels. The observed outcomes described the physiologically constrained limits. Secondly, the same outcomes were measured when using a standard operating procedure (SOP) for comparison with the determined ranges. RESULTS: The standard method resulted in similar development rates to those of high and medium diets, wing length between those resulting from the high and medium diets, and larval survival exceeding all benchmark diet level values. The SOP used to produce experimental material was shown to produces high-quality material, relative to the biologically constrained limits. CONCLUSIONS: The comparison between all possible phenotypic outcomes, as determined by biological constraints, with those outcomes obtained using a given rearing protocol is termed “benchmarking”. A method is here demonstrated which could be easily adapted to other arthropods, to objectively assess important characters obtained, and methods used, during routine culture that may affect outcomes of research. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12936-016-1288-4) contains supplementary material, which is available to authorized users

Benchmarking vector arthropod culture: an example using the African malaria mosquito, Anopheles gambiae (Diptera: Culicidae)

Background: Numerous important characteristics of adult arthropods are related to their size; this is influenced by conditions experienced as immatures. Arthropods cultured in the laboratory for research, or mass-reared for novel control methods, must therefore be of a standard size range and known quality so that results are reproducible.Methods: A simple two-step technique to assess laboratory culture methods was demonstrated using the mosquito Anopheles gambiae s.s. as a model. First, the ranges of key development outcomes were determined using various diet levels. The observed outcomes described the physiologically constrained limits. Secondly, the same outcomes were measured when using a standard operating procedure (SOP) for comparison with the determined ranges.Results: The standard method resulted in similar development rates to those of high and medium diets, wing length between those resulting from the high and medium diets, and larval survival exceeding all benchmark diet level values. The SOP used to produce experimental material was shown to produces high-quality material, relative to the biologically constrained limits.Conclusions: The comparison between all possible phenotypic outcomes, as determined by biological constraints, with those outcomes obtained using a given rearing protocol is termed “benchmarking”. A method is here demonstrated which could be easily adapted to other arthropods, to objectively assess important characters obtained, and methods used, during routine culture that may affect outcomes of research

The use of sequential mark-release-recapture experiments to estimate population size, survival and dispersal of male mosquitoes of the Anopheles gambiae complex in Bana, a west African humid savannah village

BACKGROUND: Vector control is a major component of the malaria control strategy. The increasing spread of insecticide resistance has encouraged the development of new tools such as genetic control which use releases of modified male mosquitoes. The use of male mosquitoes as part of a control strategy requires an improved understanding of male mosquito biology, including the factors influencing their survival and dispersal, as well as the ability to accurately estimate the size of a target mosquito population. This study was designed to determine the seasonal variation in population size via repeated mark-release-recapture experiments and to estimate the survival and dispersal of male mosquitoes of the Anopheles gambiae complex in a small west African village. METHODS: Mark-release-recapture experiments were carried out in Bana Village over two consecutive years, during the wet and the dry seasons. For each experiment, around 5000 (3407-5273) adult male Anopheles coluzzii mosquitoes were marked using three different colour dye powders (red, blue and green) and released in three different locations in the village (centre, edge and outside). Mosquitoes were recaptured at sites spread over the village for seven consecutive days following the releases. Three different capture methods were used: clay pots, pyrethroid spray catches and swarm sampling. RESULTS: Swarm sampling was the most productive method for recapturing male mosquitoes in the field. Population size and survival were estimated by Bayesian analyses of the Fisher-Ford model, revealing an about 10-fold increase in population size estimates between the end of dry season (10,000-50,000) to the wet season (100,000-500,000). There were no detectable seasonal effects on mosquito survival, suggesting that factors other than weather may play an important role. Mosquito dispersal ranged from 40 to 549 m over the seven days of each study and was not influenced by the season, but mainly by the release location, which explained more than 44% of the variance in net dispersal distance. CONCLUSION: This study clearly shows that male-based MRR experiments can be used to estimate some parameters of wild male populations such as population size, survival, and dispersal and to estimate the spatial patterns of movement in a given locality

Plasmodium falciparum (Haemosporodia: Plasmodiidae) and O'nyong-nyong virus development in a transgenic Anopheles gambiae (Diptera: Culicidae) strain

Transgenic Anopheles gambiae Giles (Diptera: Culicidae) mosquitoes have been developed that confer sexual sterility on males that carry a transgene encoding a protein which cuts ribosomal DNA. A relevant risk concern with transgenic mosquitoes is that their capacity to transmit known pathogens could be greater than the unmodified form. In this study, the ability to develop two human pathogens in these transgenic mosquitoes carrying a homing endonuclease which is expressed in the testes was compared with its nontransgenic siblings. Infections were performed with Plasmodium falciparum (Welch) and o'nyong-nyong virus (ONNV) and the results between the transgenic and nontransgenic sibling females were compared. There was no difference observed with ONNV isolate SG650 in intrathoracic infections or the 50% oral infectious dose measured at 14 d postinfection or in mean body titers. Some significant differences were observed for leg titers at the medium and highest doses for those individuals in which virus titer could be detected. No consistent difference was observed between the transgenic and nontransgenic comparator females in their ability to develop P. falciparum NF54 strain parasites. This particular transgene caused no significant effect in the ability of mosquitoes to become infected by these two pathogens in this genetic background. These results are discussed in the context of risk to human health if these transgenic individuals were present in the environment

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 3 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

Seasonal malaria vector and transmission dynamics in western Burkina Faso

BACKGROUND In the context of widespread mosquito resistance to currently available pesticides, novel, precise genetic vector control methods aimed at population suppression or trait replacement are a potentially powerful approach that could complement existing malaria elimination interventions. Such methods require knowledge of vector population composition, dynamics, behaviour and role in transmission. Here were characterized these parameters in three representative villages, Bana, Pala and Souroukoudingan, of the Sudano-Sahelian belt of Burkina Faso, a region where bed net campaigns have recently intensified. METHODS From July 2012 to November 2015, adult mosquitoes were collected monthly using pyrethroid spray catches (PSC) and human landing catches (HLC) in each village. Larval habitat prospections assessed breeding sites abundance at each site. Mosquitoes collected by PSC were identified morphologically, and then by molecular technique to species where required, to reveal the seasonal dynamics of local vectors. Monthly entomological inoculation rates (EIR) that reflect malaria transmission dynamics were estimated by combining the HLC data with mosquito sporozoite infection rates (SIR) identified through ELISA-CSP. Finally, population and EIR fluctuations were fit to locally-collected rainfall data to highlight the strong seasonal determinants of mosquito abundance and malaria transmission in this region. RESULTS The principal malaria vectors found were in the Anopheles gambiae complex. Mosquito abundance peaked during the rainy season, but there was variation in vector species composition between villages. Mean survey HLC and SIR were similar across villages and ranged from 18 to 48 mosquitoes/person/night and from 3.1 to 6.6% prevalence. The resulting monthly EIRs were extremely high during the rainy season (0.91-2.35 infectious bites/person/day) but decreased substantially in the dry season (0.03-0.22). Vector and malaria transmission dynamics generally tracked seasonal rainfall variations, and the highest mosquito abundances and EIRs occurred in the rainy season. However, despite low residual mosquito populations, mosquitoes infected with malaria parasites remained present in the dry season. CONCLUSION These results highlight the important vector control challenge facing countries with high EIR despite the recent campaigns of bed net distribution. As demonstrated in these villages, malaria transmission is sustained for large parts of the year by a very high vector abundance and high sporozoite prevalence, resulting in seasonal patterns of hyper and hypo-endemicity. There is, therefore, an urgent need for additional vector control tools that can target endo and exophillic mosquito populations

Studies of transgenic mosquitoes in disease-endemic countries: preparation of containment facilities

Novel approaches to area-wide control of vector species offer promise as additional tools in the fight against vectored diseases. Evaluation of transgenic insect strains aimed at field population control in disease-endemic countries may involve international partnerships and should be done in a stepwise approach, starting with studies in containment facilities. The preparations of both new-build and renovated facilities are described, including working with local and national regulations regarding land use, construction, and biosafety requirements, as well as international guidance to fill any gaps in regulation. The examples given are for containment categorization at Arthropod Containment Level 2 for initial facility design, classification of wastes, and precautions during shipping. Specific lessons were derived from preparations to evaluate transgenic (non-gene drive) mosquitoes in West and East African countries. Documented procedures and the use of a non-transgenic training strain for trial shipments and culturing were used to develop competence and confidence among the African facility staff, and along the chain of custody for transport. This practical description is offered to support other research consortia or institutions preparing containment facilities and operating procedures in conditions where research on transgenic insects is at an early stage

Conceptual framework and rationale

The sterile insect technique (SIT) has been shown to be an effective and sustainable genetic approach to control populations of selected major pest insects, when part of area-wide integrated pest management (AW-IPM) programmes. The technique introduces genetic sterility in females of the target population in the field following their mating with released sterile males. This process results in population reduction or elimination via embryo lethality caused by dominant lethal mutations induced in sperm of the released males. In the past, several field trials have been carried out for mosquitoes with varying degrees of success. New technology and experience gained with other species of insect pests has encouraged a reassessment of the use of the sterility principle as part of integrated control of malaria vectors. Significant technical and logistic hurdles will need to be overcome to develop the technology and make it effective to suppress selected vector populations, and its application will probably be limited to specific ecological situations. Using sterile males to control mosquito vector populations can only be effective as part of an AW-IPM programme. The area-wide concept entails the targeting of the total mosquito population within a defined area. It requires, therefore, a thorough understanding of the target pest population biology especially as regards mating behaviour, population dynamics, dispersal and level of reproductive isolation. The key challenges for success are: 1) devising methods to monitor vector populations and measuring competitiveness of sterile males in the field, 2) designing mass rearing, sterilization and release strategies that maintain competitiveness of the sterile male mosquitoes, 3) developing methods to separate sexes in order to release only male mosquitoes and 4) adapting suppression measures and release rates to take into account the high reproductive rate of mosquitoes. Finally, success in area-wide implementation in the field can only be achieved if close attention is paid to political, socio-economic and environmental sensitivities and an efficient management organization is established taking into account the interests of all potential stakeholders of an AW-IPM programme

Towards a sterile insect technique field release of Anopheles arabiensis mosquitoes in Sudan: Irradiation, transportation, and field cage experimentation

<p>Abstract</p> <p>Background</p> <p>The work described in this article forms part of a study to suppress a population of the malaria vector <it>Anopheles arabiensis </it>in Northern State, Sudan, with the Sterile Insect Technique. No data have previously been collected on the irradiation and transportation of anopheline mosquitoes in Africa, and the first series of attempts to do this in Sudan are reported here. In addition, experiments in a large field cage under near-natural conditions are described.</p> <p>Methods</p> <p>Mosquitoes were irradiated in Khartoum and transported as adults by air to the field site earmarked for future releases (400 km from the laboratory). The field cage was prepared for experiments by creating resting sites with favourable conditions. The mating and survival of (irradiated) laboratory males and field-collected males was studied in the field cage, and two small-scale competition experiments were performed.</p> <p>Results</p> <p>Minor problems were experienced with the irradiation of insects, mostly associated with the absence of a rearing facility in close proximity to the irradiation source. The small-scale transportation of adult mosquitoes to the release site resulted in minimal mortality (< 6%). Experiments in the field cage showed that mating occurred in high frequencies (i.e. an average of 60% insemination of females after one or two nights of mating), and laboratory reared males (i.e. sixty generations) were able to inseminate wild females at rates comparable to wild males. Based on wing length data, there was no size preference of males for mates. Survival of mosquitoes from the cage, based on recapture after mating, was satisfactory and approximately 60% of the insects were recaptured after one night. Only limited information on male competitiveness was obtained due to problems associated with individual egg laying of small numbers of wild females.</p> <p>Conclusion</p> <p>It is concluded that although conditions are challenging, there are no major obstacles associated with the small-scale irradiation and transportation of insects in the current setting. The field cage is suitable for experiments and studies to test the competitiveness of irradiated males can be pursued. The scaling up of procedures to accommodate much larger numbers of insects needed for a release is the next challenge and recommendations to further implementation of this genetic control strategy are presented.</p