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Am. J. Trop. Med. Hyg., 68(1), 2003, pp. 1-5
Copyright © 2003 by The American Society of Tropical Medicine and Hygiene

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

FRÉDÉRIC TRIPET, YEYA T. TOURÉ, GUIMOGO DOLO, AND GREGORY C. LANZARO
Department of Pathology and Center for Tropical Diseases, University of Texas Medical Branch, Galveston, Texas; Département d’ Epidémiologie des Affections Parasitaires, Ecole Nationale de Médecine et de Pharmacie, Bamako, Mali


ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We investigated the frequencies of single and multiple matings in field-collected female Anopheles gambiae 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 females 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.


INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The extent to which females of medically important vectors species are monoandrous (inseminated by a single male) or polyandrous (inseminated by more than one male) is a fundamental aspect of their biology. The frequency of insemination may be a key element in determining the success of vector control programs. In programs involving the sterile male technique,1,2 the occurrence of variation in insemination frequencies in wild vector populations raises the concern that such a program may select for higher levels of polyandry in those populations. Programs aimed at spreading genes through natural populations via the release of genetically modified vectors also rely on models of population dynamics and genetics of vector populations in which the frequency of multiple inseminations may play a critical role.

Patterns of inseminations may also be important for understanding processes of reproductive isolation in vector species that exhibit genetic polymorphism in the form of chromosomal rearrangements such as those characterizing anopheline species complexes3–5 and other species complexes in the order Diptera.6 Evidence from the Culicidae, Drosophilidae, and other families suggest that male accessory gland proteins may modulate patterns of sperm use by females.7 In addition accessory gland proteins are responsible for inhibiting further mating,8,9 and stimulate ovulation and oviposition.10–12 Thus, sex-peptide divergence could play a role in the early processes of reproductive isolation between incipient species and data on the extent of their cross-reactivity may shed light on mechanisms of underlying speciation in these taxa.9,13

In this study, we investigated insemination patterns in 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 laboratory colonies or focused on field collected material. Inferences made from laboratory observations are limited due to the fact that mosquitoes are kept at high densities in small enclosures thus creating artificial environmental conditions that can lead to a higher rate of multiple inseminations. Field studies, on the other hand, usually made use of genetic markers to detect if more than one male sired the progeny of wild caught females. This approach is obviously much sounder and, provided that it is combined with large sample sizes, provides reliable estimates of the frequency of multiply-sired egg batches. Whether conducted with laboratory or with field-collected material all studies made to date estimated polyandry from a single egg-batch and did not consider the females’ lifetime reproduction. Thus, these estimates are indirect estimates of polyandry and may be prone to various biases.

Here we assessed the level of polyandry in An. gambiae by conducting molecular analyses of the sperm extracted from the spermatheca of females collected from a natural population in Mali. Three chromosomal forms of An. gambiae s.s., namely the Mopti, Bamako, and Savanna forms, commonly occur in this study area.14 In Mali, the Mopti form is characterized by the M molecular form of ribosomal DNA intergenic transcribed spacer (rDNA IGS) and can be distinguished from the other two forms that exhibit the S-form of rDNA IGS by a polymerase chain reaction (PCR) assay.15,16 We used a PCR to amplify microsatellite DNA sequences at four polymorphic microsatellite loci to identify sperm extracts in which more than two alleles per loci were present. Such events are indicative of the presence of sperm from more than one male and provided us with a reliable estimate of insemination frequencies in a wild An. gambiae population. Next, we combined our microsatellite DNA analyses with previous data on the IGS rDNA molecular type17 to compare insemination patterns within and between the Mopti form and the other chromosomal forms of An gambiae s.s. This information is critical to understand and distinguish the mechanisms and function of multiple inseminations within taxa as opposed to those involving cross-matings between taxa. In such events, multiple insemination may be associated with processes of reproductive isolation between forms undergoing speciation in sympatry.14,18,19


MATERIAL AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Anopheles arabiensis and An. gambiae female mosquitoes were collected daily from human habitations in the village of N’gabacoro Droit near Bamako in Mali (12°41'16'N, 7°50'20'W). These collections were made during the second week of September 1999. Previous records14 and preliminary work at that site indicated that at that time of the year the Mopti and Bamako forms dominate, with the Savanna form present at low frequency. Mosquitoes were temporarily stored in small, netted cardboard containers until being transported to the laboratory at the Malaria Research and Training Center, National School of Medicine and Pharmacology (Bamako, Mali). Upon arrival at the laboratory, female mosquitoes were killed by freezing and placed singly in 1.5-ml tubes containing 70% ethanol. They were later shipped to the University of Texas Medical Branch (Galveston, TX), where all genetic analyses were conducted.

Extraction of DNA and species/form diagnosis. Individual female mosquitoes were dissected to separate their head and thorax from the abdomen. Extraction of DNA20,21 was conducted on the female tissues and the sperm content of their spermatheca as described by Tripet and others.17 A first PCR-based diagnostic that allowed discrimination between extracts from An. arabiensis and An. gambiae22 was conducted for each sample. A second PCR was used to identify the rDNA IGS form of all female tissue and corresponding sperm extract15 (see Tripet and others17 for details).

Microsatellite DNA analysis. Primer sequences and PCR conditions for loci AG2H60, AG3H88, AG3H128, and AG3H158 were used as described by Zheng and others23 These loci were selected because previous studies had found them to be highly polymorphic in An. gambiae populations from Mali.24,25 Microsatellites were amplified by a PCR using fluorescent-labeled primers. The PCR products were fractionated by electrophoresis on high-resolution polyacrylamide gels and scanned using an ABI-377 automated sequencer and GeneScan software (Applied Biosystems, Foster City, CA). Genotyping of individual mosquitoes was then performed using the Genotyper software package (Applied Biosystems). Initially, each spermatheca extract was analyzed for the four microsatellite loci. Those samples in which multiple insemination was suspected (e.g., possessed more than two alleles at least at one locus) were further analyzed to rule out contamination with maternal DNA. This was achieved by analyzing samples from the associated head and thorax for those samples.


RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Of the 329 females identified as An. gambiae, seven had damaged spermathecae and 20 were not inseminated (7%). Of the 302 females from which sperm was recovered, 251 were successfully analyzed (83.6%) with regard to their rDNA IGS type and the rDNA type of the sperm content of their spermatheca. Microsatellites were successfully amplified from the female and sperm extracts in 237 of those cases.

Insemination patterns. Analyses of sperm DNA extracts using microsatellite markers at four loci revealed polyandry in six of 237 samples examined (2.5% of all matings) (Table 1Go). One sperm DNA extract was found contaminated by female DNA (Table 1Go). The form specific rDNA IGS diagnostic showed that of the six cases in which double-mating occurred, four cases were females of the M rDNA IGS form that mated twice with M form males (2.2% of M form matings). There were no cases of polyandry within the S form females sampled (0%). Thus, within-form multiple matings for both forms combined accounted for 1.7% of all matings. Cross-mating between forms was involved in the remaining two cases of polyandry. One was an M form female double-mated with a male of each form (0.5% of M form matings) and one was an S form female double-mated with males of each form (2% of S form matings). We also found one cross-mating between an M form female and an S form male (Table 2Go).


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TABLE 1
Microsatellite analysis and molecular-form diagnostic of maternal tissue (head and thorax) and sperm DNA in cases where sperm DNA yielded more than two alleles for at least one of the loci examined*
 

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TABLE 2
Frequency of single insemination and multiple insemination in female Anopheles gambiae
 

DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The 1.7% of polyandry within-form reported here are in agreement with the low frequencies of multiply-sired progeny estimated in previous studies of field-collected anopheline species (Table 3Go). Giglioli and Mason26 in a survey of mating plugs found very rare double plugs, indicating multiple mating in field-collected female An. melas (Table 3Go). Mating plugs are produced by males and contain accessory gland products that render females refractory to subsequent matings.8,27,28 They also act as a physical barrier deterring other males from coupling, and decreasing their chances of successful insemination until females become refractory to further mating.26,29 Because mating plugs dissolve within a couple of days after mating,30 they only allow the detection of a fraction of multiple matings. Cytogenetic and genetic studies of the progeny of wild-caught anopheline females essentially found polyandry to be extremely rare or nonexistent (Table 3Go). One possible exception is the result presented by Scarpassa and others31 in their study of the progeny of An. nunestovari females. However, due to the small number of offspring genotypes used to infer the likelihood of single or double mating in that study, those results are likely to be due to a statistical artifact. Studies based on laboratory colonies consistently showed much higher levels of polyandry, lending credence to the notion that confinement and high mosquito densities create environmental conditions that favor multiple inseminations (Table 3Go). Field observations of An. gambiae swarms indicate that under natural conditions, swarms are mostly composed of males, while females enter the swarms, mate, and leave.32 Although capable of mating more than five times within a short period of time, male anopheline mosquitoes can only produce two mating plugs within that same time period.26,33 In the laboratory, those two factors may interact to increase the likelihood that females are inseminated by a second male before they become refractory to subsequent mating. The lower proportion of males to females compared with natural conditions results in males mating so frequently that they deplete the contents of their accessory gland and cannot induce refractoriness in the females with which they mate.34,35 It is also possible that females who would otherwise leave the proximity of males after a first insemination36 are unable to do so, and this increases their chances of coupling several times in a short lapse of time. Consequently, estimates of polyandry from laboratory studies are biased and do not reflect what occurs under natural conditions. In addition to those considerations, methodologic problems associated with some genetic markers may further bias these estimates. The exceptionally high estimate of polyandry found by Goma37 using two strains differing in their insecticide susceptibility was later attributed to the instability of this marker and difficulties associated with scoring heterozygous individuals.38


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TABLE 3
Levels of polyandry in anopheline mosquitoes as reported from the literature
 
Although the approach used here represents a considerable improvement over previous methods (see the Introduction), it may not be totally flawless. For one thing, the PCR is a competitive and exponential process and if two males contribute unequally to the sperm content of a spermatheca, the sperm from the male contributing least might remain undetected. At least two studies have examined this eventuality by mixing the sperm of two males in different ratios, extracting DNA from the mixture, and amplifying microsatellite DNA sequences by the PCR. In both studies, the investigators found the method to be reliable up to a 1–10 ratio of the sperm contributions.39,40 Since small proportions of sperm contributions may sometimes remain undetected, the sperm analysis method may slightly underestimate true levels of polyandry. The precision of this method also depends on the number of loci used and their level of polymorphism. If few loci are used and/or their level of polymorphism is too low, a certain proportion of multiple inseminations may remain unnoticed because different males might exhibit similar genotypes. In this study, we used four loci exhibiting 17, 22, 28, and 16 alleles, respectively. In theory and based on male genotypes for each locus, the male genotype that would combine the four most common per-locus genotype would have a likelihood of 5 x 10-4 of occurring. Under such conditions, the probability for a double-mating involving two males with undistinguishable genotypes to occur in our study was expected to be extremely small.

The importance of cross-matings and mixed M/S double-matings in terms of potential gene flow between the chromosomal forms of An gambiae has already been discussed by Tripet and others.17 What is striking in our results is the occurrence of double-mating in two of the three cases of cross-mating. When dissecting females, we also recorded the size of the female blood meal (small, medium, or large) and whether females were semi-gravid and gravid (Tripet F, unpublished data). In the third case of cross-mating found in this study, an M form female was mated to an S form male. This female was not gravid and had a small amount of blood in her midgut. Small blood meal characterized 11 of 20 females found un-mated (55%), but only nine of 302 mated females (3%) (chi-square P < 0.001), thereby suggesting that the wrongly mated female may have behaved like she was unmated. Thus, despite the small number of cross-matings observed, they could be consistent with a scenario in which females, when mated with a male of a different chromosomal or molecular form, would mate a second time. This would also imply that the sex peptide responsible for rendering females refractory to subsequent mating does not cross-react between the M and the S molecular forms. Chromosomal/molecular forms of An. gambiae have been found largely genetically undifferentiated25 except for loci located in the ribosomal DNA area on the X chromosome.15,41,42 The patterns of insemination described here suggest that chromosomal forms may have diverged genetically with regard to some of their accessory-gland proteins. These findings are in conflict with a recent study based on implants of male accessory gland substances that dismissed a role for sex peptides in triggering female sexual refractoriness in An. gambiae.43 However, earlier work based on normal and forced copulations has unambiguously showed the importance of male accessory gland products for sexual refractoriness in An. gambiae44,45 and other anopheline species.46 Although more research is clearly warranted before any conclusion can be reached, this study suggests that in addition to assortative mating,17 genetic differences in accessory gland products may be responsible for restricting gene flow between chromosomal forms of An. gambiae. It is presently unknown if there are physiologic mechanisms in place that allow females to use sperm from the two males differentially or if double-mating would simply result in diluting out the wrong sperm, and thus the fitness costs of hybridization.

It is noteworthy that the patterns of sperm use proposed here are based on data from an area where the Mopti and Bamako chromosomal forms dominated, and it is unknown if the same considerations could also apply to matings involving Mopti and Savanna or Savanna and Bamako and Savanna individuals. Cytogenetic studies of polytene chromosomes found no hybrid or backcross-like inversion arrangements indicative of introgression between Mopti and Bamako, while they were relatively common for the other two combinations of forms. Larvae exhibiting hybrid rDNA IGS genotypes have been regularly found in an area where the Mopti, Bamako, and Savanna forms co-occur.47 However, since no markers are available for differentiating Bamako from Savanna individuals, it is not possible to know whether those larvae and the hybrid female reported by Tripet and others17 were the progeny of Mopti and Bamako or Mopti and Savanna individuals. Further studies conducted in areas where different combinations of only two forms co-occur in sympatry might help resolve this question. They may also help evaluate the importance of seasonal changes in the forms frequencies and other factors that could potentially affect the mating behavior of An. gambiae and the likelihood of cross-mating to occur.

Received January 14, 2002. Accepted for publication April 10, 2002.

Acknowledgments: We thank D. Elnaiem, D. Norris, and two anonymous referees for comments on the manuscript.

Financial support: The research reported here was supported by a Swiss National Science Foundation fellowship # 823A-061233 to Frédéric Tripet and NIH grant AI-40306 to Gregory C. Lanzaro.

Reprint requests: Frédéric Tripet, Dept. of Entomology, University of California Davis, Davis, CA 95616, Telephone: 530-752-5833, Fax: 530-752-1537, E-mail: ftripet{at}ucdavis.edu, frtripet{at}yahoo.com

Authors’ addresses: Frédéric Tripet and Gregory C. Lanzaro, Dept. of Entomology, University of California Davis, Davis, CA 95616. Yeya T. Touré and Guimogo Dolo, Département d’ Epidémiologie des Affections Parasitaires, Ecole Nationale de Médecine et de Pharmacie, Bamako, BP 1805, Mali.


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