Laboratory rearing of Anopheles arabiensis: impact on genetic variability and implications for Sterile Insect Technique (SIT) based mosquito control in northern Sudan.

BACKGROUND: Mosquito colony populations often show significant changes in their population genetic make-up compared to the field populations that were used as founding source. Most of the changes that have been reported are indicators of depletion in the overall genetic diversity of the colony populations. The Sterile Insect Techniques programme of mosquito control that is underway in Northern Sudan uses sterilized males produced from a laboratory-maintained colony population. The genetic diversity of an advanced generation of this colony population was quantitatively assessed and compared to the field population from which the colony was derived. METHODS: Anopheles arabiensis mosquito samples from the 13th generation of the colony, and from the locality that was the source of the first generation of the colony, were genotyped at 11 microsatellite loci distributed throughout the species' genome. Standard population genetic analyses were carried out to quantify and compare their population genetic make-up and diversities. RESULTS: The colony samples showed significant reduction in the total number of alleles, the numbers of rare and private alleles, and the fractions of heterozygote individuals at all the loci. The pattern of change is consistent with the expected effect of the use of a small number of mosquitoes when the colony was established. Departure from Hardy-Weinberg equilibrium in the direction of homozygote excess was observed at some loci and attributed to the presence of null-alleles. CONCLUSIONS: This study highlights the need for broad sampling when initiating colony populations and for ongoing assessment of the population genetic make-up of colony populations. Previous assessments of survivorship, dispersive behaviour and swarm formation indicate that the inbreeding and reduced genetic variability reported in this study may not have had direct fitness consequences yet. However, noting the lessons learned in other SIT programmes about the impact of colonization on male sexual behaviour and longevity, as well as other inbreeding related adverse effects, a systematic investigation of these potential effects is recommended because they have direct impact on the ultimate success of the programme.

Genetic sex separation of the malaria vector, Anopheles arabiensis, by exposing eggs to dieldrin.

BACKGROUND: The sterile insect technique (SIT) has been used with success for suppressing or eliminating important insect pests of agricultural or veterinary importance. In order to develop SIT for mosquitoes, female elimination prior to release is essential as they are the disease-transmitting sex. A genetic sexing strain (GSS) of Anopheles arabiensis was created based on resistance to dieldrin, and methods of sex separation at the egg stage were developed. The use of this strain for SIT will require sexually sterile males: useful radiation doses for this purpose were determined for pupae and adults. METHODS: For the creation of the sexing strain, dieldrin-resistant males were irradiated with 40 Gy using a 60Co source and were subsequently crossed to homozygous susceptible virgin females. Individual families were screened for semi-sterility and for male resistance to dieldrin. For sex separation, eggs of a resulting GSS, ANO IPCL1, were exposed to varying concentrations of dieldrin for different durations. Percent hatch, larval survival, and male and female emergence were recorded. Radiation induced sterility was determined following adult and pupa exposure to gamma rays at 0-105 Gy. Mortality induced by dieldrin treatment, and levels of sterility post radiation were investigated. RESULTS: ANO IPCL1 contains a complex chromosome aberration that pseudo-links the male-determining Y chromosome and dieldrin resistance, conferring high natural semi-sterility. Exposure of eggs to 2, 3, and 4 ppm dieldrin solutions resulted in complete female elimination without a significant decrease of male emergence compared to the controls. A dose of 75 Gy reduced the fertility to 3.8 and 6.9% when males were irradiated as pupae or adults respectively, but the proportions of progeny of these males reaching adulthood were 0.6 and 1.5% respectively CONCLUSION: The GSS ANO IPCL1 was shown to be a suitable strain for further testing for SIT though high semi-sterility is a disadvantage for mass rearing.

Ethical, legal and social aspects of the approach in Sudan.

The global malaria situation, especially in Africa, and the problems frequently encountered in chemical control of vectors such as insecticide resistance, emphasize the urgency of research, development and implementation of new vector control technologies that are applicable at regional and local levels. The successful application of the sterile insect technique (SIT) for the control of the New World screwworm Cochliomyia hominivorax and several species of fruit flies has given impetus to the use of this method for suppression or elimination of malaria vectors in some areas of Africa including Northern State of Sudan. The research and development phase of the Northern State feasibility study has been started. Sudanese stakeholders are working side-by-side with the International Atomic Energy Agency in the activities of this important phase. Several ethical, legal and social issues associated with this approach arose during this phase of the project. They need to be seriously considered and handled with care. In this paper, these issues are described, and the current and proposed activities to overcome potential hurdles to ensure success of the project are listed.

Sex separation strategies: past experience and new approaches.

The success of the sterile insect technique (SIT) and other genetic strategies designed to eliminate large populations of insects relies on the efficient inundative releases of competitive, sterile males into the natural habitat of the target species. As released sterile females do not contribute to the sterility in the field population, systems for the efficient mass production and separation of males from females are needed. For vector species like mosquitoes, in which only females bite and transmit diseases, the thorough removal of females before release while leaving males competent to mate is a stringent prerequisite. Biological, genetic and transgenic approaches have been developed that permit efficient male-female separation for some species considered for SIT. However, most sex separation methods have drawbacks and many of these methods are not directly transferable to mosquitoes. Unlike genetic and transgenic systems, biological methods that rely on sexually dimorphic characters, such as size or development rate, are subject to natural variation, requiring regular adjustment and re-calibration of the sorting systems used. The yield can be improved with the optimization of rearing, but the scale of mass production places practical limits on what is achievable, resulting in a poor rearing to output ratio. High throughput separation is best achieved with scalable genetic or transgenic approaches.

Field site selection: getting it right first time around.

The selection of suitable field sites for integrated control of Anopheles mosquitoes using the sterile insect technique (SIT) requires consideration of the full gamut of factors facing most proposed control strategies, but four criteria identify an ideal site: 1) a single malaria vector, 2) an unstructured, relatively low density target population, 3) isolation of the target population and 4) actual or potential malaria incidence. Such a site can exist in a diverse range of situations or can be created. Two contrasting SIT field sites are examined here: the desert-flanked Dongola Reach of the Nile River in Northern State, Sudan, where malaria is endemic, and the island of La Reunion, where autochthonous malaria is rare but risk is persistent. The single malaria-transmitting vector at both sites is Anopheles arabiensis. In Sudan, the target area is a narrow 500 km corridor stretching from the rocky terrain at the Fourth Cataract--just above the new Merowe Dam, to the northernmost edge of the species range, close to Egypt. Vector distribution and temporal changes in density depend on the Nile level, ambient temperature and human activities. On La Reunion, the An. arabiensis population is coastal, limited and divided into three areas by altitude and exposure to the trade winds on the east coast. Mosquito vectors for other diseases are an issue at both sites, but of primary importance on La Reunion due to the recent chikungunya epidemic. The similarities and differences between these two sites in terms of suitability are discussed in the context of area-wide integrated vector management incorporating the SIT.

Spatial and temporal distribution of the malaria mosquito Anopheles arabiensis in northern Sudan: influence of environmental factors and implications for vector control.

BACKGROUND: Malaria is an important public health problem in northern Sudan, but little is known about the dynamics of its transmission. Given the characteristic low densities of Anopheles arabiensis and the difficult terrain in this area, future vector control strategies are likely to be based on area-wide integrated pest management (AW-IPM) that may include the sterile insect technique (SIT). To support the planning and implementation of future AW-IPM activities, larval surveys were carried out to provide key data on spatial and seasonal dynamics of local vector populations. METHODS: Monthly cross-sectional larval surveys were carried out between March 2005 and May 2007 in two localities (Dongola and Merowe) adjacent to the river Nile. A stratified random sampling strategy based on the use of Remote Sensing (RS), Geographical Information Systems (GIS) and the Global Positioning System (GPS) was used to select survey locations. Breeding sites were mapped using GPS and data on larval density and breeding site characteristics were recorded using handheld computers. Bivariate and multivariate logistic regression models were used to identify breeding site characteristics associated with increased risk of presence of larvae. Seasonal patterns in the proportion of breeding sites positive for larvae were compared visually to contemporaneous data on climate and river height. RESULTS: Of a total of 3,349 aquatic habitats sampled, 321 (9.6%) contained An. arabiensis larvae. The frequency with which larvae were found varied markedly by habitat type. Although most positive sites were associated with temporary standing water around the margins of the main Nile channel, larvae were also found at brickworks and in areas of leaking pipes and canals - often far from the river. Close to the Nile channel, a distinct seasonal pattern in larval populations was evident and appeared to be linked to the rise and fall of the river level. These patterns were not evident in vector populations breeding in artificial water sources away from the river. CONCLUSION: The GIS-based survey strategy developed in this study provides key data on the population dynamics of An. arabiensis in Northern State. Quantitative estimates of the contributions of various habitat types and their proximity to settlements provide a basis for planning a strategy for reducing malaria risk by elimination of the vector population.

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

BACKGROUND: The work described in this article forms part of a study to suppress a population of the malaria vector Anopheles arabiensis 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

The origin and evolution of mosquito APE retroposons.

The detection of horizontal transfer is important to understanding the origin and spread of transposable elements and in assessing their impact on genetic diversity. The occurrence of the phenomenon is not in doubt for two of the three major groups of elements, but is disputed for retroposons, largely on the grounds of data paucity and overreliance on divergence estimates between host species. We present here the most wide-ranging retroposon data set assembled to date for a species group, the mosquitoes. The results provide no evidence for horizontal transfer events and show conclusively that four previously reported events, involving Juan-A, Juan-C, T1, and Q, did not occur. We propose that the origin of all known mosquito retroposons can be attributed to vertical inheritance and that retroposons have therefore been a persistent source of genetic diversity in mosquito genomes since the emergence of the taxon. Furthermore, the data confirm that the unprecedented levels of retroposon diversity previously reported in Anopheles gambiae extends to at least seven other species representing five genera and all three mosquito subfamilies. Most notably, this included the L1 elements, which are not known in other insects. A number of novel well-defined monophyletic groups were also identified, particularly, JM2 and JM3 within the Jockey clade, which included sequences from seven and five mosquito species, respectively. As JM3 does not contain an Anopheles element, this represents a good example of stochastic loss and the best out of at least four found in this study. This exceptionally diverse data set when compared with the wealth of data available for the many unrelated species with which mosquitoes have intimate contact through blood feeding ought to be fertile ground for the discovery of horizontal transfer events. The absence of positive results therefore supports the view that retroposon horizontal transfer does not occur or is far more exceptional than for other types of transposable elements.

Emerging vectors in the Culex pipiens complex.

In the Old World, some mosquitoes in the Culex pipiens complex are excellent enzootic vectors of West Nile virus, circulating the virus among birds, whereas others bite mainly humans and other mammals. Here we show that, in northern Europe, such forms differing in behavior and physiology have unique microsatellite fingerprints with no evidence of gene flow between them, as would be expected from distinct species. In the United States, however, hybrids between these forms are ubiquitous. Such hybrids between human-biters and bird-biters may be the bridge vectors contributing to the unprecedented severity and range of the West Nile virus epidemic in North America.