Identification of field caught Anopheles gambiae s.s. and Anopheles arabiensis by TaqMan single nucleotide polymorphism genotyping

BACKGROUND: Identification of Anopheles gambiae s.s. and Anopheles arabiensis from field-collected Anopheles gambiae s.l. is often necessary in basic and applied research, and in operational control programmes. The currently accepted method involves use of standard polymerase chain reaction amplification of ribosomal DNA (rDNA) from the 3' 28S to 5' intergenic spacer region of the genome, and visual confirmation of amplicons of predicted size on agarose gels, after electrophoresis. This report describes development and evaluation of an automated, quantitative PCR method based upon TaqMan™ single nucleotide polymorphism (SNP) genotyping. METHODS: Standard PCR, and TaqMan SNP genotyping with newly designed primers and fluorophore-labeled probes hybridizing to sequences of complementary rDNA specific for either An. gambiae s.s. or An. arabiensis, were conducted in three experiments involving field-collected An. gambiae s.l. from western Kenya, and defined laboratory strains. DNA extraction was from a single leg, sonicated for five minutes in buffer in wells of 96-well PCR plates. RESULTS: TaqMan SNP genotyping showed a reaction success rate, sensitivity, and species specificity comparable to that of standard PCR. In an extensive field study, only 29 of 3,041 (0.95%) were determined to be hybrids by TaqMan (i.e., having rDNA sequences from both species), however, all but one were An. arabiensis by standard PCR, suggesting an acceptably low (ca. 1%) error rate for TaqMan genotyping in mistakenly identifying species hybrids. CONCLUSION: TaqMan SNP genotyping proved to be a sensitive and rapid method for identification of An. gambiae s.l. and An. arabiensis, with a high success rate, specific results, and congruence with the standard PCR method

Principal component analysis of socioeconomic factors and their association with malaria in children from the Ashanti Region, Ghana

<p>Abstract</p> <p>Background</p> <p>The socioeconomic and sociodemographic situation are important components for the design and assessment of malaria control measures. In malaria endemic areas, however, valid classification of socioeconomic factors is difficult due to the lack of standardized tax and income data. The objective of this study was to quantify household socioeconomic levels using principal component analyses (PCA) to a set of indicator variables and to use a classification scheme for the multivariate analysis of children < 15 years of age presented with and without malaria to an outpatient department of a rural hospital.</p> <p>Methods</p> <p>In total, 1,496 children presenting to the hospital were examined for malaria parasites and interviewed with a standardized questionnaire. The information of eleven indicators of the family's housing situation was reduced by PCA to a socioeconomic score, which was then classified into three socioeconomic status (poor, average and rich). Their influence on the malaria occurrence was analysed together with malaria risk co-factors, such as sex, parent's educational and ethnic background, number of children living in a household, applied malaria protection measures, place of residence and age of the child and the mother.</p> <p>Results</p> <p>The multivariate regression analysis demonstrated that the proportion of children with malaria decreased with increasing socioeconomic status as classified by PCA (p < 0.05). Other independent factors for malaria risk were the use of malaria protection measures (p < 0.05), the place of residence (p < 0.05), and the age of the child (p < 0.05).</p> <p>Conclusions</p> <p>The socioeconomic situation is significantly associated with malaria even in holoendemic rural areas where economic differences are not much pronounced. Valid classification of the socioeconomic level is crucial to be considered as confounder in intervention trials and in the planning of malaria control measures.</p

Survival of immature Anopheles arabiensis (Diptera: Culicidae) in aquatic habitats in Mwea rice irrigation scheme, central Kenya

BACKGROUND: The survivorship and distribution of Anopheles arabiensis larvae and pupae was examined in a rice agro-ecosystem in Mwea Irrigation Scheme, central Kenya, from August 2005 to April 2006, prior to implementation of larval control programme. METHODS: Horizontal life tables were constructed for immatures in semi-field condition. The time spent in the various immature stages was determined and survival established. Vertical life tables were obtained from five paddies sampled by standard dipping technique. RESULTS: Pre-adult developmental time for An. arabiensis in the trays in the experimental set up in the screen house was 11.85 days from eclosion to emergence. The mean duration of each instar stage was estimated to be 1.40 days for first instars, 2.90 days for second instars, 1.85 days for third instars, 3.80 days for fourth instars and 1.90 days for pupae. A total of 590 individuals emerged into adults, giving an overall survivorship from L1 to adult emergence of 69.4%. A total of 4,956 An. arabiensis immatures were collected in 1,400 dips throughout the sampling period. Of these, 55.9% were collected during the tillering stage, 42.5% during the transplanting period and 1.6% during the land preparation stage. There was a significant difference in the An. arabiensis larval densities among the five stages. Also there was significant variation in immature stage composition for each day's collection in each paddy. These results indicate that the survival of the immatures was higher in some paddies than others. The mortality rate during the transplanting was 99.9% and at tillering was 96.6%, while the overall mortality was 98.3%. CONCLUSION: The survival of An. arabiensis immatures was better during the tillering stage of rice growth. Further the survival of immatures in rice fields is influenced by the rice agronomic activities including addition of nitrogenous fertilizers and pesticides. For effective integrated vector management, the application of larvicides should target An. arabiensis larvae at the tillering stage (early vegetative stage of rice) when their survival in the aquatic habitats is high to significantly reduce them and the larvicides should be long-lasting to have a significant impact on the malaria vector productivity on the habitats

Spatial and temporal distribution patterns of Anopheles arabiensis breeding sites in La Reunion Island - multi-year trend analysis of historical records from 1996-2009

<p>Abstract</p> <p>Background</p> <p>An often confounding facet of the dynamics of malaria vectors is the aquatic larval habitat availability and suitable conditions under which they can thrive. Here, we investigated the impact of environmental factors on the temporal and spatial distribution of larval habitats of <it>Anopheles </it><it>arabiensis </it>in different locations on La Reunion Island.</p> <p>Methods</p> <p>A retrospective examination was made from archival data which provided the complete enumeration of <it>An. arabiensis </it>breeding habitats in three distinct geographic zones - extending North-east, West and South of the island over 14 years, from January 1996 to December 2009. Data on the occurrence and the number of active larval habitats at each of a total of 4376 adjacent ellipsoid grid cells (216,506 square meters each) were used (1) to provide the geographic extent of breeding site availability from year to year and (2) to analyze associations with prevailing environmental factors, habitat types, and locations.</p> <p>Results</p> <p><it>Anopheles arabiensis </it>utilized a spectrum of man-made and natural aquatic habitats, most of which were concentrated primarily in the rock pools located in ravines and river fringes, and also in the large littoral marshes and within the irrigated agricultural zones. The numbers of breeding site per sampling grid differed significantly in different parts of the island. In contrast to an originally more widespread distribution across the island in the 1950s, detailed geographic analyses of the data obtained in the period extending from 1996-2009 showed an intriguing clustered distribution of active breeding sites in three discontinuous geographic zones, in which aquatic habitats availability fluctuates with the season and year. Seasonality in the prevalence of anopheles breeding sites suggests significant responsiveness to climatic factors.</p> <p>Conclusions</p> <p>The observed retreat of <it>An. arabiensis </it>distribution range to lower altitudinal zones (< 400 m) and the upward shift in the most remote littoral areas in the northeast and southwest regions suggest the possible influence of biogeographic factors, changes in land use and control operations. The results of this study would allow for a more rational implementation of control strategies across the island.</p

Unexpected High Losses of Anopheles gambiae Larvae Due to Rainfall

Background - Immature stages of the malaria mosquito Anopheles gambiae experience high mortality, but its cause is poorly understood. Here we study the impact of rainfall, one of the abiotic factors to which the immatures are frequently exposed, on their mortality. Methodology/Principal Findings - We show that rainfall significantly affected larval mosquitoes by flushing them out of their aquatic habitat and killing them. Outdoor experiments under natural conditions in Kenya revealed that the additional nightly loss of larvae caused by rainfall was on average 17.5% for the youngest (L1) larvae and 4.8% for the oldest (L4) larvae; an additional 10.5% (increase from 0.9 to 11.4%) of the L1 larvae and 3.3% (from 0.1 to 3.4%) of the L4 larvae were flushed away and larval mortality increased by 6.9% (from 4.6 to 11.5%) and 1.5% (from 4.1 to 5.6%) for L1 and L4 larvae, respectively, compared to nights without rain. On rainy nights, 1.3% and 0.7% of L1 and L4 larvae, respectively, were lost due to ejection from the breeding site. Conclusions/Significance - This study demonstrates that immature populations of malaria mosquitoes suffer high losses during rainfall events. As these populations are likely to experience several rain showers during their lifespan, rainfall will have a profound effect on the productivity of mosquito breeding sites and, as a result, on the transmission of malaria. These findings are discussed in the light of malaria risk and changing rainfall patterns in response to climate chang

Spatial Analysis of Land Cover Determinants of Malaria Incidence in the Ashanti Region, Ghana

Malaria belongs to the infectious diseases with the highest morbidity and mortality worldwide. As a vector-borne disease malaria distribution is strongly influenced by environmental factors. The aim of this study was to investigate the association between malaria risk and different land cover classes by using high-resolution multispectral Ikonos images and Poisson regression analyses. The association of malaria incidence with land cover around 12 villages in the Ashanti Region, Ghana, was assessed in 1,988 children <15 years of age. The median malaria incidence was 85.7 per 1,000 inhabitants and year (range 28.4–272.7). Swampy areas and banana/plantain production in the proximity of villages were strong predictors of a high malaria incidence. An increase of 10% of swampy area coverage in the 2 km radius around a village led to a 43% higher incidence (relative risk [RR] = 1.43, p<0.001). Each 10% increase of area with banana/plantain production around a village tripled the risk for malaria (RR = 3.25, p<0.001). An increase in forested area of 10% was associated with a 47% decrease of malaria incidence (RR = 0.53, p = 0.029)

Evidence of Introgression of the ace-1R Mutation and of the ace-1 Duplication in West African Anopheles gambiae s. s

Background: The role of inter-specific hybridisation is of particular importance in mosquito disease vectors for predicting the evolution of insecticide resistance. Two molecular forms of Anopheles gambiae s.s., currently recognized as S and M taxa, are considered to be incipient sibling species. Hybrid scarcity in the field was suggested that differentiation of M and S taxa is maintained by limited or absent gene flow. However, recent studies have revealed shared polymorphisms within the M and S forms, and a better understanding of the occurrence of gene flow is needed. One such shared polymorphism is the G119S mutation in the ace-1 gene (which is responsible for insecticide resistance); this mutation has been described in both the M and S forms of A. gambiae s.s. Methods and Results: To establish whether the G119S mutation has arisen independently in each form or by genetic introgression, we analysed coding and non-coding sequences of ace-1 alleles in M and S mosquitoes from representative field populations. Our data revealed many polymorphic sites shared by S and M forms, but no diversity was associated with the G119S mutation. These results indicate that the G119S mutation was a unique event and that genetic introgression explains the observed distribution of the G119S mutation within the two forms. However, it was impossible to determine from our data whether the mutation occurred first in the S form or in the M form. Unexpectedly, sequence analysis of some resistant individuals revealed a duplication of the ace-1 gene that was observed in both A. gambiae s.s. M and S forms. Again, the distribution of this duplication in the two forms most likely occurred through introgression. Conclusions: These results highlight the need for more research to understand the forces driving the evolution of insecticide resistance in malaria vectors and to regularly monitor resistance in mosquito populations of Africa

Behavioural responses of Anopheles gambiae sensu stricto M and S molecular form larvae to an aquatic predator in Burkina Faso

Background: Predation of aquatic immature stages has been identified as a major evolutionary force driving habitat segregation and niche partitioning in the malaria mosquito Anopheles gambiae sensu stricto in the humid savannahs of Burkina Faso, West Africa. Here, we explored behavioural responses to the presence of a predator in wild populations of the M and S molecular forms of An. gambiae that typically breed in permanent (e.g., rice field paddies) and temporary (e.g., road ruts) water collections. Methods: Larvae used in these experiments were obtained from eggs laid by wild female An. gambiae collected from two localities in south-western Burkina Faso during the 2008 rainy season. Single larvae were observed in an experimental arena, and behavioural traits were recorded and quantified a) in the absence of a predator and b) in the presence of a widespread mosquito predator, the backswimmer Anisops jaczewskii. Differences in the proportion of time allocated to each behaviour were assessed using Principal Component Analysis and Multivariate Analysis of Variance. Results: The behaviour of M and S form larvae was found to differ significantly; although both forms mainly foraged at the water surface, spending 60-90% of their time filtering water at the surface or along the wall of the container, M form larvae spent on average significantly more time browsing at the bottom of the container than S form larvae (4.5 vs. 1.3% of their overall time, respectively; P < 0.05). In the presence of a predator, larvae of both forms modified their behaviour, spending significantly more time resting along the container wall (P < 0.001). This change in behaviour was at least twice as great in the M form (from 38.6 to 66.6% of the time at the wall in the absence and presence of the predator, respectively) than in the S form (from 48.3 to 64.1%). Thrashing at the water surface exposed larvae to a significantly greater risk of predation by the notonectid (P < 0.01), whereas predation occurred significantly less often when larvae were at the container wall (P < 0.05) and might reflect predator vigilance. Conclusions: Behavioural differences between larvae of the M and S form of An. gambiae in response to an acute predation risk is likely to be a reflection of different trade-offs between foraging and predator vigilance that might be of adaptive value in contrasting aquatic ecosystems. Future studies should explore the relevance of these findings under the wide range of natural settings where both forms co-exist in Africa