- Open Access
Behavioural determinants of gene flow in malaria vector populations: Anopheles gambiae males select large females as mates
© Okanda et al; licensee BioMed Central Ltd. 2002
- Received: 7 June 2002
- Accepted: 14 August 2002
- Published: 14 August 2002
Plasmodium-refractory mosquitoes are being rapidly developed for malaria control but will only succeed if they can successfully compete for mates when released into the wild. Pre-copulatory behavioural traits maintain genetic population structure in wild mosquito populations and mating barriers have foiled previous attempts to control malaria vectors through sterile male release.
Varying numbers of virgin male and female Anopheles gambiae Giles, from two strains of different innate sizes, were allowed to mate under standardized conditions in laboratory cages, following which, the insemination status, oviposition success and egg batch size of each female was assessed. The influence of male and female numbers, strain combination and female size were determined using logistic regression, correlation analysis and a simple mechanistic model of male competition for females.
Male An. gambiae select females on the basis of size because of much greater fecundity among large females. Even under conditions where large numbers of males must compete for a smaller number of females, the largest females are more likely to become inseminated, to successfully oviposit and to produce large egg batches.
Sexual selection, on the basis of size, could either promote or limit the spread of malaria-refractory genes into wild populations and needs to be considered in the continued development and eventual release of transgenic vectors. Fundamental studies of behavioural ecology in malaria vectors such as An. gambiae can have important implications for malaria control and should be prioritised for more extensive investigation in the future.
- Malaria Control
- Large Female
- Female Size
- Strain Combination
Malaria remains one of world's most burdensome and intractable diseases [1–3]. As the Roll Back Malaria campaign proceeds to promote the effective implementation of existing control methods, one of the most rapidly developing new strategies is the possibility of rendering wild vector populations less susceptible to infection by releasing genetically-modified mosquitoes [4–7]. Transposable genetic elements may allow relatively small releases of such transgenic laboratory-reared mosquitoes to drive Plasmodium-refractory genes to fixation in wild vector populations, even if these genes confer substantially reduced fitness [8, 9]. However, the fixation of malaria-refractory genes will ultimately depend upon the ability of the laboratory-reared transgenics to survive and mate with wild vectors following their release . Recent studies have also shown that the fitness costs of insecticide resistance genes in mosquitoes can include reduced mating competitiveness among males . Pre-copulatory behavioural barriers are therefore not only important because of their influence upon the genetic structure of wild vector populations  but also our future ability to prevent these insects from spreading malaria . Recently, the utility of transgenic mosquitoes as a malaria control tool has been seriously questioned and the ecology of targeted vector species has been identified as a poorly understood topic which needs to be investigated in much more detail before any such releases can be envisaged in the future [5, 7, 8].
Unfortunately most investigations of mating behaviour in mosquitoes have concentrated on species that are active during the day  whereas the important vectors of malaria are predominantly crepuscular or nocturnal species such as An. gambiae Giles . We have therefore quantified determinants of mating and oviposition success in An.gambiae, one of the world's most effective vectors of malaria [14, 15] and found that males compete aggressively for selected females based on the size of the latter.
The bulk of this work was carried out using the Ifakara strain of An. gambiae, originally colonized from wild caught gravid females in the Kilombero valley, Tanzania. For the cross-strain mating experiments, the Mbita strain, similarly colonized in Western Kenya were used and both strains were reared under semi-natural conditions in separate screen-walled greenhouses exposed to ambient airflow and meteorological conditions on the shores of Lake Victoria, in Western Kenya [16–18]. All experiments were conducted in 30 cm cubic netting cages within additional screen-walled greenhouses where females were placed in paper cups within cages containing males 1 hr before release. The mosquitoes were released and allowed to interact within the cage between 1600–0800 h. For experiments to identify determinants of insemination success and fecundity, all females were offered bloodmeals on three consecutive days and then individually transferred to separate glass vials with a paper strip to rest on, 6% glucose and an oviposition pad. These mosquitoes were then allowed five days to oviposit following which the insemination status of non-ovipositing females determined by dissection and examination of the spermathecae
Mechanistic modeling of mating success as a function of competition
Firstly, it was assumed that all females are receptive to insemination but that the ability of males to inseminate females depends on whether other competing males are present. We define these abilities in terms of the rate at which single and additional competing males inseminate individual females, denotes I1 and Ic. We assume that a single male inseminates a given, limited proportion of females and that insemination of remaining females by additional competing males progresses at a rate proportional to the number of additional males (M). Assuming that, for all experiments, the amount of time allowed for mating was constant (one night), and applying a standard first order decay function, the proportion remaining uninseminated (U) is an exponential function of the proportion remaining uninseminated by a single male:
U = (1 - I1) exp - (Ic (M-1)) Equation 1
The proportion of females inseminated in each cage as a function of male density was fitted to this equation separately for each number of females, pooling all 3 replicates to give 18 observations. This nonlinear regression was applied using the NLIN procedure of SAS 8.0, fitted by maximum likelihood weighted according to the binomial variance and bounding U to values between 0 and 1 as well as I1 and Ic to positive values. In order to express the level of competition under any given conditions, we define the competition index (C) as the rate at which individual males alone in a cage succeed in inseminating females divided by the rate at which those in competition with others do so:
C = I1 / Ic Equation 2
Using the Ifakara strain of An.gambiae, we found that peaks of insemination activity at dusk and dawn were associated with an elliptical flight pattern, typical of swarming dipterans , in which a number of male and female mosquitoes form a cluster on one side of the cage. Males routinely jostled with each other in flight and often displaced others from their resting positions on the netting. Male flight was very rapid with several individuals flying through the same loops, pursuing each other while facing in the same direction. It was during these interactions that males most frequently succeeded in grasping and copulating with females. The females flew much less than the males and although transient flights over short distances occasionally occurred, they were most commonly observed when grasped by males. Consistent with previous reports [19, 20], one single male was found to inseminate up to 10 females per night (Mean ± SEM = 8.3 ± 1.0, n = 4 experiments) when placed in cage with 30 of them. However, the addition of 1, 2 or 3 males (that is doubling, tripling or quadrupling the male numbers in a cage) did not increase the proportion of the 30 females that became inseminated. This suggests that males compete with each other for the same limited proportion of females rather than distributing their attention to inseminate as many females as possible.
Final fitted logistic regression model accounting for variations in probability of female insemination as a function of male and female numbers per cage and replicate. Deviance and Pearsons χ2 goodness of fit statistics were 3.35 and 3.31 per degree of freedom, respectively, with 49 degrees of freedom and P < 0.0001 for both.
β ± SEM
-0.599 ± 0.070
Reciprocal of females per cage
6.12 ± 1.79
Males per cage
0.00586 ± 0.00054
The ability of males to inseminate females as a function of competition and female density. Estimates for the proportion of females inseminated by the addition of one male only (I1) and the rate at which they are inseminated by additional competing males (Ic) were estimated by fitting a simple mechanistic model and the competition index (C) calculated as their quotient (See materials and methods).
Females per cage
0.422 ± 0.053
0.0073 ± 0.0016
58 ± 15
0.338 ± 0.042
0.0040 ± 0.0009
84 ± 21
0.362 ± 0.045
0.0031 ± 0.0009
116 ± 37
Dependence of female insemination probability and fecundity upon size, male competition level and strain.*
β ± SEM
Ifakara strain only
1 & 2
5.4 ± 1.8
-1.44 ± 0.59
-38.1 ± 10.7
13.1 ± 3.7
1.91 ± 1.0
Oviposition after insemination†
0.15 ± 0.56
-1.95 ± 0.95
All four male-female combinations of Ifakara and Mbita strains
-13.0 ± 3.4
2.37 ± 0.38
3.98 ± 1.12
-10.2 ± 3.1
1.83 ± 0.34
3.06 ± 1.01
Oviposition after insemination†
2.52 ± 0.39
Egg batch size of ovipositors‡
-1.16 ± 0.47
0.94 ± 0.15
In contrast with An. freeborni, recent field studies of An.gambiae have shown that large males are no more likely to fall out of mating swarms in copula than are small males . Here, however, we have shown that the size of female An.gambiae has major impact on not only their fecundity [22, 23] but also their chance of being chosen as mates by the males who compete intensely for the privilege. Under these laboratory conditions, female size is the major factor influencing mate choice by males within and between strains as well as the size of subsequent egg batches. The behaviour by colony-adapted mosquitoes within the confines of relatively small cages may not necessarily reflect equivalent processes in wild field populations and further investigation in larger semi-field systems [16–18, 25] and natural populations are clearly required. Although the studies we have described here do illustrate the kind of ethological determinants of gene flow that may exist in nature, further investigation in the field is essential because the process of adapting wild mosquitoes to colony rearing and maintenance inevitably selects for behaviour that is compatible with successful mating under artificially high densities within the confines of small cages. Nevertheless, our observations are fully consistent with current insect sexual selection theory, which indicates that the choosiness of males has evolved in response to strong correlation between the selected trait and fecundity of females . Such closely associated sexual and reproductive fitness traits, as well as natural selection based upon them, are therefore likely to be important in wild field populations of An.gambiae from which these captive strains were derived and may have important practical implications for malaria control in the future.
Here we have shown that the size of female An. gambiae may be a critically important determinant of their reproductive fitness in terms of their ability to compete for mates and their ability to lay large egg batches. In practical terms this may have important implications for the success of malaria control measures such as the release of genetically modified mosquitoes that are refractory to malaria because such strategies are inherently dependent upon the reproductive fitness of the released mosquitoes [8, 9]. For example, rearing conditions for breeding batches for release will need to be carefully considered because overcrowding or poor nutrition of larvae result in smaller, reproductively disadvantaged adults [22, 27]. Similarly, size heterogeneities arising from variability in larval habitat quality [28, 29] may affect gene flow patterns in the field and need to be considered when driving exogenous genes into field populations which are spatially and temporally structured . Beyond their direct implications, our observations also illustrate how fundamental studies of behavioural ecology in mosquitoes can have important implications for malaria control and should be prioritised for more extensive investigation in the future [7, 11].
We thank V. Robert and P. Billingsley for insightful comments and support from NIH through International Centre for Infectious Disease Research (U19-AI45511), Actions in Building Capacity (D43-TW01142) and Kenya-Mali Collaborative Exchange (D43 TW00920) awards.
We also thank S. Bopp for assistance with preliminary experiments and P. Ongele for technical assistance in the insectary.
- Breman JG, Egan A, Keutsch GT: The intolerable burden of malaria: a new look at the numbers. Am J Trop Med Hyg. 2001, 64 (Supplement 1): iv-vii.PubMedGoogle Scholar
- Sachs J, Malaney P: The economic and social burden of malaria. Nature. 2002, 415: 680-685. 10.1038/415680a.View ArticlePubMedGoogle Scholar
- Killeen GF, McKenzie FE, Foy BD, Schieffelin C, Billingsley PF, Beier JC: The potential impacts of integrated malaria transmission control on entomologic inoculation rate in highly endemic areas. Am J Trop Med Hyg. 2000, 62: 545-551.PubMed CentralPubMedGoogle Scholar
- Aultman KS, Beaty BJ, Walker ED: Genetically manipulated vectors of human disease: a practical overview. Trends Parasitol. 2001, 17: 507-509. 10.1016/S1471-4922(01)02094-3.View ArticlePubMedGoogle Scholar
- Spielman A, Pollack RJ, Kiswewski AE, Telford SR: Issues in Public Health Entomology. Vector Borne Zoon Dis. 2001, 1: 3-19. 10.1089/153036601750137606.View ArticleGoogle Scholar
- Ito J, Ghosh A, Moreira LA, Wimmer EA, Jacobs-Lorena M: Transgenic anopheline mosquitoes impaired in transmission of a malaria parasite. Nature. 2002, 417: 452-455. 10.1038/417452a.View ArticlePubMedGoogle Scholar
- Enserink M: Ecologists see flaws in transgenic mosquito. Science. 2002, 297: 30-31. 10.1126/science.297.5578.30b.View ArticlePubMedGoogle Scholar
- Boete C, Koella JC: A theoretical approach to predicting the success of genetic manipulation of malaria mosquitoes in malaria control. Malar J. 2002, 1: 3-10.1186/1475-2875-1-3.PubMed CentralView ArticlePubMedGoogle Scholar
- Kiszewski AE, Spielman A: Spatially explicit model of transposon-based genetic drive mechanisms for displacing fluctuating populations of anopheline vector mosquitoes. J Med Entomol. 1998, 35: 584-90.View ArticlePubMedGoogle Scholar
- Berticat C, Boquien G, Raymond M, Chevillon C: Insecticide resistance genes induce a mating competition cost in Culex pipiens. Genet Res. 2002, 79: 41-47. 10.1017/S001667230100547X.View ArticlePubMedGoogle Scholar
- Donnelly MJ, Simard F, Lehmann T: Evolutionary studies of malaria vectors. Trends Parasitol. 2002, 18: 75-80. 10.1016/S1471-4922(01)02198-5.View ArticlePubMedGoogle Scholar
- Downes JA: The swarming and mating flight of Diptera. Annu Rev Entomol. 1969, 14: 271-298. 10.1146/annurev.en.14.010169.001415.View ArticleGoogle Scholar
- Jones MDR, Gubbins SJ: Change in the ciracidian flight activity of the mosquito Anopheles gambiae in relation to insemination, feeding and oviposition. Physiol Entomol. 1978, 3: 213-220.View ArticleGoogle Scholar
- White GB: Anopheles gambiae complex and disease transmission in Africa. Trans R Soc Trop Med Hyg. 1974, 68: 278-301.View ArticlePubMedGoogle Scholar
- Beier JC, Killeen GF, Githure J: Short report: Entomologic inoculation rates and Plasmodium falciparum malaria prevalence in Africa. Am J Trop Med Hyg. 1999, 61: 109-113.PubMedGoogle Scholar
- Seyoum A, Hassanali A, Kabiru EW, Killeen GF, Luande W, Knols BGJ: Live potted plants repel Anopheles gambiae from semi-field experimental huts. Am J Trop Med Hyg. 2002,Google Scholar
- Seyoum A, Hassanali A, Kabiru EW, Palsson K, Killeen GF, Luande W, Knols BGJ: Traditional use of mosquito repellent plants in Western Kenya and their evaluation in semi-field experimental huts against Anopheles gambiae : Ethnobotanical studies and application by thermal expulsion and direct burning. Trans R Soc Trop Med Hyg. 2002, 96: 225-231.View ArticlePubMedGoogle Scholar
- Mathenge E, Killeen GF, Oulo D, Irunga L, Nfegwa P, Knols BGJ: Development of an exposure-free bednet trap for sampling Afrotropical malaria vectors. Med Vet Entomol. 2002, 16: 67-74. 10.1046/j.0269-283x.2002.00350.x.View ArticlePubMedGoogle Scholar
- Bryan JH: Further studies on consecutive matings in the Anopheles gambiae complex. Nature. 1972, 239: 519-520.View ArticlePubMedGoogle Scholar
- Goma LKH: Tests for multiple insemination in Anopheles gambiae Giles. Nature. 1963, 197: 99-100.View ArticleGoogle Scholar
- Yuval B, Wekesa JW, Washino RK: Effect of body size on swarming behaviour and mating success of male Anopheles freeborni (Diptera: Culicidae). J Insect Behav. 1993, 6: 333-342.View ArticleGoogle Scholar
- Lyimo E, Takken W: Effects of adult body size on fecundity and pre-gravid rate of Anopheles gambiae females in Tanzania. Med Vet Entomol. 1993, 7: 328-332.View ArticlePubMedGoogle Scholar
- Hogg JC, Thomson MC, Hurd H: Comparative fecundity and associated factors for two sibling species of the Anopheles gambiae complex occurring sympatrically in The Gambia. Med Vet Entomol. 1996, 10: 385-91.View ArticlePubMedGoogle Scholar
- Charlwood JD, Pinto J, Sousa CA, Ferreira C, Do Rosario VE: Male size does not affect mating success of Anopheles gambiae in Sao Tome. Med Vet Entomol. 2002, 16: 109-111. 10.1046/j.0269-283x.2002.00342.x.View ArticlePubMedGoogle Scholar
- Russell PF, Rao TR: On the swarming, mating and oviposition behaviour of Anopheles culifacies. Am J Trop Med Hyg. 1942, 22: 417-427.Google Scholar
- Bonduriansky R: The evolution of male mate choice in insects: a synthesis of ideas and evidence. Biol Rev Camb Philos Soc. 2001, 76: 305-339. 10.1017/S1464793101005693.View ArticlePubMedGoogle Scholar
- Lyimo EO, Takken W, Koella JC: Effect of rearing temperature and larval density on larval survival, age at pupation and adult size of Anopheles gambiae. Entomol Exp Appl. 1992, 63: 265-271.View ArticleGoogle Scholar
- Gimnig JE, Ombok M, Kamau L, Hawley WA: Characteristics of larval anopheline (Diptera: Culicidae) habitats in Western Kenya. J Med Entomol. 2001, 38: 282-8.View ArticlePubMedGoogle Scholar
- Gimnig JE, Ombok M, Otieno S, Kaufman MG, Vulule JM, Walker ED: Density-dependent development of Anopheles gambiae (Diptera: Culicidae) larvae in artificial habitats. J Med Entomol. 2002, 39: 162-172.View ArticlePubMedGoogle Scholar
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