Anopheles gambiae, the major malaria vector in Africa, can be divided into two subgroups based on genetic and ecological criteria. These two subgroups, termed the M and S molecular forms, are believed to be incipient species. Although they display differences in the ecological niches they occupy in the field, they are often sympatric and readily hybridize in the laboratory to produce viable and fertile offspring. Evidence for assortative mating in the field was recently reported, but the underlying mechanisms awaited discovery. We studied swarming behaviour of the molecular forms and investigated the role of swarm segregation in mediating assortative mating. Molecular identification of 1145 males collected from 68 swarms in Donéguébougou, Mali, over 2 years revealed a strict pattern of spatial segregation, resulting in almost exclusively monotypic swarms with respect to molecular form. We found evidence of clustering of swarms composed of individuals of a single molecular form within the village. Tethered M and S females were introduced into natural swarms of the M form to verify the existence of possible mate recognition operating within-swarm. Both M and S females were inseminated regardless of their form under these conditions, suggesting no within-mate recognition. We argue that our results provide evidence that swarm spatial segregation strongly contributes to reproductive isolation between the molecular forms in Mali. However this does not exclude the possibility of additional mate recognition operating across the range distribution of the forms. We discuss the importance of spatial segregation in the context of possible geographic variation in mechanisms of reproductive isolation.

The role of chromosomal inversions in speciation has long been of interest to evolutionists. Recent quantitative modeling has stimulated reconsideration of previous conceptual models for chromosomal speciation. Anopheles gambiae, the most important vector of human malaria, carries abundant chromosomal inversion polymorphism nonrandomly associated with ecotypes that mate assortatively. Here, we consider the potential role of paracentric inversions in promoting speciation in A. gambiae via ''ecotypification,'' a term that refers to differentiation arising from local adaptation. In particular, we focus on the Bamako form, an ecotype characterized by low inversion polymorphism and fixation of an inversion, 2Rj, that is very rare or absent in all other forms of A. gambiae. The Bamako form has a restricted distribution by the upper Niger River and its tributaries that is associated with a distinctive type of larval habitat, laterite rock pools, hypothesized to be its optimal breeding site. We first present computer simulations to investigate whether the population dynamics of A. gambiae are consistent with chromosomal speciation by ecotypification. The models are parameterized using field observations on the various forms of A. gambiae that exist in Mali, West Africa. We then report on the distribution of larvae of this species collected from rock pools and more characteristic breeding sites nearby. Both the simulations and field observations support the thesis that speciation by ecotypification is occurring, or has occurred, prompting consideration of Bamako as an independent species. chromosomal inversion ͉ ecological speciation ͉ Bamako chromosomal form ͉ inversion polymorphism ͉ selection

Mosquito swarms are poorly understood mating aggregations. In the malaria vector Anopheles gambiae Giles, they are known to depend on environmental conditions, such as the presence of a marker on the ground, and they may be highly relevant to reproductive isolation. We present quantitative measurements of individual An. gambiae positions within swarms from Donéguébougou, Mali, estimated by stereoscopic video image analysis. Results indicate that swarms in this species are approximately spherical, with an unexpectedly high density of individuals close to the swarm centroid. This high density may be the result of individual males maximizing their probability of encountering a female or a product of mosquito orientation through cues within the swarm. Our analysis also suggests a difference in swarm organization between putative incipient species of An. gambiae with increasing numbers of males. This may be related to a difference in marker use between these groups, supporting the hypothesis that swarming behavior is a mechanism of mate recognition and ultimately reproductive isolation. KeywordsAnopheles gambiae; swarm; mate recognition; three-dimensional localization; stereoscopic image analysis Mosquito swarms have long been observed and described, but with few exceptions (such as Gibson 1985), their physical organization and dynamics remain poorly understood, especially in the field. In this paper, we address two fundamental issues as they relate to swarms of the malaria vector Anopheles gambiae Giles. The first is how swarms of males are organized in this species. The second is what role this organization might play in mate choice and reproductive isolation within the An. gambiae species complex.Part of the reason little is known about mosquito swarms is that they are generally difficult to study in a natural setting. Locating the swarms can be difficult for many species (Service 1993), and even once found, it is difficult to gather quantitative measurements (although not impossible; see Yuval and Bouskila 1993). Previous studies on insect swarming generally used image acquisition and processing techniques to examine swarms (Okubo et al. 1981, Riley 1993. Stereoscopic image analysis in particular has been used to study mosquito swarms, resulting in some sophisticated statistical methods for localizing individuals (Ikawa and Okabe 1997). However, these approaches have yielded limited biological insight into the organization 2Corresponding author, e-mail: E-mail: manoukisn@niaid.nih.gov. Anopheles gambiae swarms are known to be composed almost entirely of males (Diabaté et al. 2006), and are often, although not always, found over "swarming markers" (Marchand 1984, Charlwood et al. 2002. As in other dipterans, they are thought to be mating aggregations (Downes 1969, Sullivan 1981. They probably fit the strict definition of a lek (an area where males congregate to secure mates), especially because they represent non-resource-based aggregations. There may be competition for more advantageous positions within t...

We describe a novel tracking system for reconstructing three-dimensional tracks of individual mosquitoes in wild swarms and present the results of validating the system by filming swarms and mating events of the malaria mosquito Anopheles gambiae in Mali. The tracking system is designed to address noisy, low frame-rate (25 frames per second) video streams from a stereo camera system. Because flying A. gambiae move at 1-4 m s 21, they appear as faded streaks in the images or sometimes do not appear at all. We provide an adaptive algorithm to search for missing streaks and a likelihood function that uses streak endpoints to extract velocity information. A modified multi-hypothesis tracker probabilistically addresses occlusions and a particle filter estimates the trajectories. The output of the tracking algorithm is a set of track segments with an average length of 0.6-1 s. The segments are verified and combined under human supervision to create individual tracks up to the duration of the video (90 s). We evaluate tracking performance using an established metric for multitarget tracking and validate the accuracy using independent stereo measurements of a single swarm. Three-dimensional reconstructions of A. gambiae swarming and mating events are presented.