FREQUENCY OF MULTIPLE INSEMINATIONS IN FIELD-COLLECTED ANOPHELES GAMBIAE FEMALES REVEALED BY DNA ANALYSIS OF TRANSFERRED SPERM

We investigated the frequencies of single and multiple matings in field-collected female Anopheles gam- biae by conducting microsatellite DNA analyses on the sperm contained within their spermatheca. Amplifcation by a polymerase chain reaction (PCR) at four loci allowed the detection of sperm extracts exhibiting more than two alleles per locus, thereby revealing the occurrence of multiple inseminations. Polyandry was found in six of 239 fen;tales examined, or 2.5% of the samples. Previous analyses of the molecular form of the sperm and female extracts using a PCR-based diagnostic procedure showed that two of these multiple inseminations involved cross-mating between two chromosomal/molecular forms of An. gambiae s.s. Thus polyandry occurred within-form in 1.7% of examined females while other multiple inseminations may be linked to processes of reproductive isolation between forms of An. gambiae. INTRODUcrION field-collected material all studies made to date estimated polyandry from a single egg-batch and did not consider the The extent to which females of medically important vectors females' lifetime reproduction. Thus, these estimates are in- species are monoandrous (inseminated by a single male) or direct estimates of polyandry and may be prone to various polyandrous (inseminated by more than one male) is a fun- biases. damental aspect of their biology. The frequency of insemina- Here we assessed the level of polyandry in An. gambiae by tion may be a key element in determining the success of vec- conducting molecular analyses of the sperm extracted from tor control programs. In programs involving the sterile male the spermatheca of females collected from a natural popula- technique!.2 the occurrence of variation in insemination fre- tion in Mali. Three chromosomal forms of An. gambiae s.s., quencies in wild vector populations raises the concern that namely the Mopti, Bamako, and Savanna forms, commonly such a program may select for higher levels of po)yandry in occur in this study area.14 In Mali, the Mopti form is charac- those populations. Programs aimed at spreading genes terized by the M molecular form of ribosomal DNA inter- through natural populations via the release of genetically genic transcribed spacer (rDNA IGS) and can be distin- modified vectors also rely on models of population dynamics guished from the other two forms that exhibit the S-form of and genetics of vector populations in which the frequency of rDNA IGS by a polymerase chain reaction (PCR) assay.1S.16 multiple inseminations may playa critical role. We used a PCR to amplify microsatellite DNA sequences at Patterns of inseminations may also be important for under- four polymorphic microsatellite loci to identify sperm extracts standing processes of reproductive isolation in vector species in which more than two alleles per loci were present. Such that exhibit genetic polymorphism in the form of chromosom- events are indicative of the presence of sperm from more than al rearrangements such as those characterizing anopheline one male and provided us with a reliable estimate of insemi- species complexes3-S and other species complexes in the or- nation frequencies in a wild An. gambiae population. Next, der Diptera.6 Evidence from the Culicidae, Drosophilidae, we combined our rnicrosatellite DNA analyses with previous and other families suggest that male accessory gland proteins data on the IGS rDNA molecular type17 to compare insemi- may modulate patterns of sp~rm use by females.7 In addition nation patterns within and between the Mopti form and the accessory gland proteins are responsible for inhibiting further other chromosomal forms of An gambiae s.s. This information mating,8.9 and stimulate ovulation and oviposition.1°-12 Thus, is critical to understand and distinguish the mechanisms and sex-peptide divergence could playa role in the early processes function of multiple inseminations within taxa as opposed to of reproductive isolation between incipient species and data those involving cross-matings between taxa. In such events, on the extent of their cross-reactivity may shed light on multiple insemination may be associated with processes of mechanisms of underlying speciation in these taxa.9.13 reproductive isolation between forms undergoing speciation In this study, we investigated insemination patterns in in sympatry.14,18.19 Anopheles gambiae s. s. Giles, the main vector of malaria in tropical Africa. Previous studies of mating patterns of anopheline mosquitoes were based on observations of labo